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AN ABSTRACT OF THE THESIS OF

Mu Wang for the degree of Master of Science in Chemistry presented on June

17. 1993.

Title: Studies on the Biosynthesis of Capreomycin

Abstract approved:

Professor Steven . Gould

Capreomycin is an antitubercular antibiotic produced by Streptomyces capreolus. It

consists of four unique structural components: IA, IB, 11.A. and IIB.

The biosynthesis of capreomycin has been studied by administration of isotopically

labeled (14C, 13C and 2H) precursors to S. capreolus A250. The results from feeding

radiolabeled compounds indicated that serine was a most likely precursor. Feedings of

stable isotopically labeled serine (10a and 10b) suggested that the 2,3-diaminopropionate

(Dap) moieties of capreomycin are derived from serine via a dehydroalanyl intermediate.

The level of enrichment at the serine residue of capreomycin IA, 1, compared to those at

the other sites of 1 and at all sites of capreomycin IB, 2, suggested that the serine residue

in 1 derives from serine directly whereas the alanine residue in 2 comes from alanine, and

this was later confirmed by feeding labeled alanine (12a). Therefore, 1 is derived

independently from 2 rather than by a hydroxylation of 2.

Syntheses and feedings of deuteriated Dap (11a and 11b) demonstrated that Dap is

an intermediate in the biosynthesis of capreomycin, and that the P-ureido-dehydroalanine

(Uda) residue is also derived from Dap, presumbly by addition of carbamoyl phosphate.

Redacted for privacy

Preliminary experiments have detected that cell-free extracts of S. capreolus contain

the enzymatic activity which in the presence of the precursor amino acids of capreomycins

catalyzes an ATP /[32P]pyrophosphate exchange reaction. The experiments suggest that the

substrates (precursor amino acids) are activated by the formation of substrate-AMP in

correspondence with a non-ribosomal "thiotemplate" mechanism of peptide biosynthesis.

The further purification and characterization of these enzyme activities are clearly of

interest.

STUDIES ON THE BIOSYNTHESIS OF CAPREOMYCIN

by

Mu Wang

A THESIS

submitted to

Oregon State University

in partial fulfillment of

the requirements for

the degree of

Master of Science

Completed June 17, 1993

Commencement June, 1994

Approved:

Professor of Chemistry in Charge o ajor

Chairman of Department of Chemistry

Date Thesis is Presented: June 17. 1993

Typed by Mu Wang for Mu Wang




To My Wife Jirong , My Son Kevin and My Parents

Bad moments, like good ones, tend to be grouped together.

Edna O'Brien

Acknowledgements

First and foremost, I am deeply indebted to my advisor, Dr. Steven J. Gould, for

his inspiring guidance and unfailing enthusiasm which made this work possible.

I also would like to thank Drs. Donna Minott and Martha Cone for their suggestions

and help when I first started my research in the "Gould Group". My special recognition

goes to Dr. Tom O'Hare, Dr. Nuria Tamayo and Marc Kirchmeier, for their advice, helpful

discussions and most of all for their friendship. Mr. Rodger Kohnert is thanked for hishelp with NMR spectroscopy.

Finally, I wish to express my gratitude to my wife Jirong, my son Kevin and my

parents for their continuous understanding and support

Table of ContentsPage

Introduction 1

Capreomycin: A Cyclic Pentapeptide Antibiotic 1

Structurally Related Peptide Antibiotics 4

Previous Biosynthetic Studies on Capreomycin 6

Objectives and Significance of Present Studies 10

Results and Discussion 13

Fermentation Study 13

Bioassay with Bacillus subtilis 13

Fermentation of S. capreolus 14

Purification of Capreomycins 16

Synthesis 17

DL- [2,3,3- 2H3]Dap (11a) 17

DL-[2-2H]Dap (11b) 18

Hydrolysis of Capreomycin and Purification of Capreomycidine (5) 19

Feeding Experiments 20

L-R1-14C1Serine, L-[U-14C]Alanine and L-[U-14C[Cystine 20

DL-[1-13C]Serine (10a) 20

DL- [3- 13C]Alanine (12a) 24

DL-[2,3,3-2H3]Serine (10b) 27

DL- [2,3,3- 2H3]Dap (11a) 30

DL- [2- 2H]Dap (11b) 30

Exchange Reaction 33

Studies with Cell-free Extract from S. capreolus 35

Preparation and Partial Purification of Cell-free Extract (CI.E) 36

Protein Concentration Determination 37

Enzyme Assay 38

Conclusions and Areas for Further Study 41

Experimental

General

44

44

Table of Contents, Continued

Page

Fermentation of S. capreolus A250 45

Maintenance of S. capreolus A250 45

Production of Capreomycins 45

Isolation of Capreomycins IA and 113 46

Bioassay 47

Synthesis 47

DL-[2,3,3-2H3]Dap (11a) 47

DL- [2- 2H]Dap (11b) 49

Hydrolysis of Capreomycin and Purification of Capreomycidine (5) 49

Feedings 50

Feeding Protocol 50

Calculations in Feeding Experiments 51

L-[U-14C]Serine, L-[U-14C]Alanine and L-[Up4qCystine 51

DL- [1- 13C]Serine (10a) 52

DL- [3- 13C]Alanine (12a) 52

DL-[2,3,3-2H3]Serine (10b) 53

DL-[2,3,3-2H3]Dap (11a) 53

DL- [2- 2H]Dap (11b) 53

Exchange Reaction of Capreomycin 1B under the Fermentation Conditions 54

Cell-free Extract from S. capreolus 55

Preparation and Partial Purification of Cell-free Extract from S. capreolus 55

Protein Concentration Determination 56

Enzyme Activity Assays 56

Bibliography 58

List of Figures

Figure Page

1. Structure of Capreomycins 1

2. Schematic View of Viomycin and Capreomycins 2

3. Proton and Carbon Assignments of Capreomycin IA 3

4. Proton and Carbon Assignments of Capreomycin IB 4

5. Structure of Tuberactinomycins 5

6. Structure of LL-BM547r3 and LL-BM547a 6

7. Proposed Biogenesis of the Streptolidine Moiety of Streptothricin F (7) 7

8. Proposed Biogenesis of Capreomycidine 8

9. Scheme of Ribosomal (a) and Non-ribosomal (b) Biosynthesis of Peptide

Antibiotics 12

10. Dose-dependent Inhibition of B. subtilis Growth by Capreomycin

Concentration Dependence 14

11. Production Curve of Capreomycins 16

12. Typical Chromatogram of Capreomycins 17

13a. Enrichments over Natural Abundance Observed in lb 21

13b. Enrichments over Natural Abundance Observed in 2b 22

14. Proposed Metabolic Pathways for Incorporation of [1-13C]Serine into

r3-Lysine, Capreomycidine and p-Ureido-dehydroalanine 23

15a. Enrichments over Natural Abundance Observed in lc 24

15b. Enrichments over Natural Abundance Observed in 2c 25

16. Proposed Metabolic Pathway for [3-13C]Alanine Feeding 26

17. NMR Spectra of 2d 28

18. NMR Spectra of ld 29

19. Partial NMR Spectra of le and if 31

20. Partial NMR Spectra of 2e and 2f 32

21. Summary of Labeling Patterns of 1 and 2 from Feeding

Experiments with Precursor Amino Acids 34

22. Bradford Assay: BSA Standards 37

23. The Apparent Manner of Capreomycin Assembly 42

List of Tables

Page

Table 1 Results of Radioactive Precursor Feedings 20

Table 2 Specific Activity of [32P]ATP from ATP/[32P]PPi

Exchange Reaction 40

STUDIES ON THE BIOSYNTHESIS OF CAPREOMYCIN

Introduction

Capreomycin: A Cyclic Pentapeptide Antibiotic

The antitubercular antibiotic capreomycin, a metabolite of Streptomyces

capreolus, was first isolated by Herr, et al. in 1960.1 It was subsequently reported to be

composed of two components I and H in 1962,2 and in 1965 was finally shown to

actually contain four components: IA, re, HA and BB (1, 2, 3, and 4, respectively) as

shown in Figure 1.3

RO

H2N14eN

ONH HN

N N NH2

0NH

N

1: R = OH, R' f3- Lysine2: R = H, R' = f3- Lysine3: R = OH, R' =H4: R = H, R' = H

Figure 1. Structure of Capreomycins

In general, capreomycin, which resembles viomycin4 chemically and

pharmacologically, is a second-line agent employed in combination with other

antitubercular drugs. It is active against a number of Gram-positive and Gram-negative

bacteria, but is primarily effective against mycobacteria. In particular, it may be used in

2

place of standard antitubercular drugs such as streptomycin and rifampicin where either

the patient is sensitive to, or the strain of Mycobacterium tuberculosis is resistant to

streptomycin or rifampicin.5,6 The structures of capreomycins were proposed by a

Japanese group in l976,7 but was later revised 8,9 It was concluded that the amino acid

sequence in the cyclic peptide moiety corresponded to that of viomycin. However, the

position linking the branched amino acid, 3- lysine, was surprisingly found to differ from

that of viomycin (Figure 2). When capreomycin was dinitrophenylated and subsequently

hydrolyzed, Na-dinitrophenyl-diaminopropionate (Dap) but not Ni-dinitrophenyl-

diaminopropionate was detected.

Da Pt

Dap1

Sere Sera

Tbd5 Uda4

Viomycin

Dap3 13-Lys

Cpd 5 Uda4

X

Capreomycin IA : SerineCapreomycin IB : Alanine

Abbreviations: Tbd: L-tuberactidine; Dap: 2,3-diaminopropionic acid;

Cpd: L-capreomycidine; Uda: 13-ureido-dehydroalanine.

Figure 2. Schematic View of Viomycin and Capreomycins

3

Detailed examinations of NMR spectra of capreomycins, particularly for the a-

amide and a-methine protons of Dapi and 0-amide proton of Dap3, as well as Edman-

dansyl degradation on reduced capreomycin IA, strongly supported the structure depicted

in Figure 1, in which the 0-lysine residue is linked to the 0-amino group of the Dap3

residue.10 Total syntheses of capreomycins IA and IB have been reported," and

complete assignments of 1H and 13C NMR spectra of both capreomycins IA and IB

(Figure 3 and Figure 4) have been carried out using one and two dimensional NMR

spectroscopy.12

4.41

H2 H

52.37

3.87 HOH

63.80

168.63 56.20

H3.24 40.89

4.1H 0NH

172.52

0 56.60

4.53

1.74

2.12 24.35

3.33

Hsig

4.843.80 337

NH2 1.82 1.82

H31530.48

173 53 4983 24.36

H H3.11

173.7

30 55.04

173.13

0

10621168 79 NH2

10 136.70 14

39.40

H 4.44H

172.83 2.69

1.82 1.82

50.48 Q

NH

37.49 7 155.87

NH2

H 8.05 0

Figure 3. Proton and Carbon Assignments of Capreomycin IA

NH2

H2

19.63CH3 144

324.68

H

3.86 3.71O

3.29 40.96

H ,, ----

4.15n NH H( H 0

1 172.40lisl

H157.92 NH2

H 5.00 10 136.71 14

2.81 2.691.82 1.82

1.71

2.09 24.24

3.34HH

Figure 4. Proton and Carbon Assignments of Capreomycin IB

NH2

4

Structurally Related Peptide Antibiotics

Additional members of this group, called tuberactinomycins (Figure 5), have

subsequently been characterized from other species. Although they are produced by

different species or genera of bacteria, the common structural pattern among these

antibiotics suggests that they are biogenetically related.

H2N

NH2 0N Tr

ONH u/HN

0"N

HN

H

0NH

R2 N NH2H

OH

0

NH2

0

Tuberactinomycin R1 R2

A OH OHB (Viomycin) H OHN OH HO H H

Figure 5. Structure of Tuberactinomycins

5

Tuberactinomycin was initially isolated from fermentation broths of Streptomyces

griseoverticillatus var. tuberacticus, which had been found in the soil at Ohitocho near

Mount Fuji, Japan, by Nagata et al. of the Toyo Jozo Company in 1968.13 The original

strain produced tuberactinomycins A and B, which were separated by ion exchange

chromatography. The chromatographic properties of tuberactinomycin B were found to

be identical to those of the known antibiotic viomycin, whose structure had not been

established at the time.14 From a mutant obtained by nitrosoguanidine treatment of the

original strain, new congeners N and 0 were later isolated.14-16 All congeners of

tuberactinomycin exhibit remarkable and comparable activities against, in particular,

Mycobacterium and Corynebacterium.13-16 Commercially available enviomycin,17

which is used as an antitubercular agent, is composed mainly of tuberactinomycin N.

6

Other antitubercular peptide antibiotics, LL-BM54713 and LL-BM547a (Figure

6), were isolated from a Nocardia species, Leder le culture BM547. Studies against M.

tuberculosis in mice showed LL -BM547 j3 to be as effective as either viomycin or

streptomycin. However, the tolerated dose of LL-BM54713 was 32 times less than that of

streptomycin and 16 times less than that of viomycin. The biological activity of LL-

BM547a is less than that of LL-BM54713 or viomycin.16

NH

HO NNH

LL-BM54713: R =110-CH3-13-arginyl

LL-BM547a: R = H

Figure 6. Structure of LL-BM547r3 and LL-BM547a

Previous Biosynthetic Studies on Capreomycin

The biochemical origins of the residues that constitute capreomycins have

received little attention. The first biosynthetic study of capreomycin was recently carried

out in our research group,18 and focused on L-capreomycidine (5), the most unusual

amino acid component in the capreomycins. The synthesis of capreomycidine has been

reported.19 Its biosynthetic origin was proposed20 to be as an intermediate in the

biosynthesis of streptolidine (6),21 a key component of streptothricin antibiotics,22,23

such as streptothricin F (7, Figure 7) .24,25

OH H

H

Ne NHH H II

0

H2N

COOH

9

0II

H2NCOOH

H

5

HO HN NH2

0 NH2

7

7

Figure 7.18 Proposed Biogenesis of the Streptolidine Moiety (6) [shown in box]

of Streptothricin F (7)

The biosynthesis of 7 has been studied extensively in our group,26-31 and the

intermediacy of 5 was fully consistent with results from the incorporation of a series of

15N-13C-labeled arginines.27,28 However, in further probing the mechanism of formation

of the cyclic guanidine ring of the putative capreomycidine moiety, unusual results were

obtained with a variety of arginines labeled with deuterium at H-2 and/or H-3.28 When

the pentadeuteriated arginine (8a) was fed to a fermentation culture of Streptomyces L-

1689-23,31 loss of deuterium from both H-2 and H-3 was observed in the product thus

obtained, 7a (Scheme 1). This is inconsistent with the original proposal involving a,0-

dehydroarginine (9, Figure 8, pathway a),21 but could still be rationalized by a pathway

via 3-hydroxy- or 3-keto-arginine (Figure 8, pathway b or c).28

8

H2N

H

H2N7 COOH

8

1H2N

H2N COOH

N

0A COOH

H2NOH

H2NH COOH

H2N

H2N

9

Scheme 1

HN NH

HN

H2N COOH

5

Figure 8. Proposed Biogenesis of Capreomycidi

HN. ID

D

H

00HC HD

8a

S. L-1689-23

7a

9

However, when both erythro- and threo-3-hydroxyarginines were synthesized and

tested as intermediates the results were negative,31 neither being incorporated nor

apparently produced de novo. Therefore, the biosynthesis of 5 in S. capreolus became

significant in terms of mechanistic studies of the formation of capreomycidine.

The investigation of the biosynthesis of 5 in S. capreolus involved feeding both

14C-labeled arginine and 2H-labeled arginine to fermentations of S. capreolus and

determining the 14C incorporation and 2H enrichment in isolated capreomycins by liquid

scintillation counting and 2H NMR spectroscopy, respectively. When L-RJ-14C]arginine

was fed, it was incorporated into both IA (8.5% incorporation) and IB (12.7%

incorporation), suggesting that arginine is one of the primary precursors for the

biosynthesis of capreomycins. In order to obtain positional information, a sample of the

deuteriated arginine (8a) was next fed, and the samples of capreomycins IA and IB, la

and 2a, respectively, derived from 8a were analyzed by 2H NMR spectroscopy. The

results, shown in Scheme 2, clearly revealed the retention of one deuterium atom at H-3

and two deuterium atoms at H-5 but not at H-2.

Scheme 2

COOHH H 0

8a la, 2a

S. capreolusH

This result suggested that the capreomycidine (5) residue is derived from arginine

(8) via an a,13-dehydroarginyl intermediate (Figure 8, pathway a). Since the manner of

incorporation of 8a into the capreomycins is apparently quite different than into

streptothricin F, this study did not shed light on the latter process. However, the

10

capreomycin results are readily explained by intramolecular addition of the guanidine to

an a,13-dehydroarginyl residue. Since in the capreomycins capreomycidine is actually

found as part of a peptide structure, and N-acyl-a,13-dehydroamino acids are stable, it is

reasonable to assume that the capreomycidine ring arises from a post-peptide assembly

modification of arginine in a peptide intermediate.

Objectives and Significance of Present Studies

The present studies mainly focused on determining the origins of the remaining

four amino acid residues of the cyclic pentapeptide cores of 1 and 2. The Dap and Uda

residues represent non-protein amino acids. The Dap residue, which is also found in

malonomicin (active against Trypanosoma congolense),32 L-N3-oxalo-Dap (a neurotoxin

responsible for neurolathyrism),33,34 and the tuberactinomycin35 and bleomycin36

families (antituberculosis and antitumor antibiotics, respectively), was proposed to come

from serine via a dehydroalanyl intermediate (Scheme 3).37 We recognized that two

other potential precursors, alanine and cysteine, should also be tested. The Uda residue

may be formed from addition of a guanido-group from arginine to a dehydroalanyl

residue followed by another dehydrogenation (Scheme 4, pathway a), or by addition of an

ureido-group to a Dap followed by another dehydrogenation (Scheme 4, pathway b).

Scheme 3

COOH

10

OH

"-H20" "NH3"

Scheme 4

NH2

11

N)LNH2 4..

NH2

Since 1 and 2 differ only in the presence or absence of a hydroxyl at C-32, it was

possible that 1 was derived by hydroxylation of 2 rather than by substitution of serine for

alanine in the biosynthesis. In an attempt to test this in the previous study,1 8

capreomycin IB obtained from the DL-[l -14C]arginine feeding was fed to a new

fermentation of S. capreolus. However, very little capreomycin IA, with no radioactivity,

was obtained. From this result, it was not clear whether IA is produced independently

from IB or that the administered IB could not enter the cell due to impermeability to the

(potentially toxic) compound. This needed to be further investigated. In addition,

although arginine was found to be the precursor of capreomycidine (5), it was still not

clear whether 5 arises from a pre- or post-peptide assembly modification of arginine. An

in vivo test of labeled 5 would make the whole picture clearer.

After the biosynthetic origins of the four core amino acids in the capreomycins

were determined, the next objective was to investigate the mechanism of the formation of

the peptide. Although a few examples of ribosomal biosynthesis of secondary metabolic

12

peptides have been reported,38 essentially all microbial peptide antibiotics so far studied

at the cell-free level have been shown to be formed by a non-ribosomal mechanism.39,40

Figure 9 summarizes both ribosomal and multienzymatic (non-ribosomal) processes of

peptide biosynthesis.39

In the ribosomal system, amino acids are activated by aminoacyl-tRNA

synthetases. These are symbolized here by their different types of subunit structures.

Each charged tRNA is then lined up at the ribosome according to the mRNA sequence.

The non-ribosomal biosynthesis is not really as well understood. Schematically, in this

system polyenzymes activate amino and hydroxy acids, and the template itself is

aminoacylated, generally, although not exclusively, as a thioester. A transport system

then assembles the lined-up residues, presumably by a sequential mechanism.39 The

latter, which involves multifunctional/multienzyme complexes, may be involved in the

biosynthesis of capreomycins. Thus, the capreomycins present an opportunity to study the

mechanisms and relationships of extensive pre- and post-peptide assembly modification,

in addition to the peptide assembly process itself. Our first target at the cell-free level

focused on finding constituent amino acid activating enzyme(s).

a.

b.

thioester

9s'

Arrinoacyl-tRNA synthetases

transportprotein

Figure 9.39 Scheme of Ribosomal (a) and Non-ribosomal (b) Biosynthesis of Peptide

Antibiotics

Results and Discussion

Fermentation Study

13

The fermentation of S. capreolus and the bioassay conditions, as well as

purification methods for capreomycins had been worked out by Dr. Donna A. Minott in

our laboratory. These procedures18 were utilized, with a few modifications, in all of the

feeding experiments.

Bioassay with Bacillus subtilis

Bioassay with B. subtilis ATCC 6633 was found to be sufficiently sensitive (MIC

3.13 tig/mL) for our purposes and much simpler than the previously reported

turbidometric assay.2 Prior to the bioassay, PA-7 agar (0.5% Bacto-tryptone + 1.5%

Bacto-agar) was inoculated with appropriate amounts of a diluted B. subtilis ATCC 6633

saline suspension. Sterile stainless steel wells were used as reservoirs for the antibiotic

solution. After 12 h of incubation at 37 0C, clear, distinct zones of inhibition had

appeared. A standard bioassay curve was obtained by using different concentrations of

commercially available capreomycin sulfate (Sigma). Plotting the log2 of concentration

versue the diameter of the inhibition zone gave the line shown in Figure 10.

14

20

19

18

17

16

15

14

13

12

11

10

9

8

7

61 2 3 4 5 6 7 8 9 10 11 12 13 14

Log 2 Concentration (pg/mL)

Figure 10. Dose-dependent Inhibition of B. subtilis Growth by Capreomycin

Concentration Dependence

Fermentation of S. capreolus

Fermentation conditions for S. capreolus had been tested by Dr. Donna A. Minott

in our laboratory, and the complex production medium2 was found to be suitable for our

needs. The recipes for the seed medium41 and the production medium are listed below.

15

Seed medium:

Tryptone 0.30 g

Yeast extract 0.15 g

Glucose 0.90 g

MgSO4.7H20 0.18 g

dd. H2O 60 mL (in 250 mL Erlenmeyer flask)

The pH of the solution, initially 6.8, was adjusted to 7.5 using 2% KOH before

sterilization.

Production medium:

Glucose 5.60 g

Bacto-peptone 3.00 g

N-Z Amine A 0.80 g

Blackstrap molasses 2.00 g

Starch (potato) 2.00 g

CaCO3 0.40 g

MgSO4.7H20 1.00 g

Hot tap water 200 mL (in 1000 mL Erlenmeyer flask)

The pH (6.5) was not adjusted.

Generally, the production of capreomycins (IA and IB) was in the range of 2-3

mg/mL as determined by bioassay, of which 20-40% (approximately equal amounts of

IA and IB) was recoverable by the isolation procedure. A study was performed to monitor

the production of capreomycins as a function of time (Figure 11).

50

40

E-a)E

0

maz 20

10

1:1I I I § . t

0 24 48 72 96 120 144 168 192

Time (hrs)

Figure 11. Production Curve of Capreomycins

16

Purification of Capreomycins

After 4 days of incubation (30 °C, 250 rpm), the production of capreomycins

reached a maximum (Figure 11), and the fermentation broth was typically collected after

6 days by filtration to remove the mycelia. Activated charcoal was then added to the

filtrate to bind all of the organic materials. After filtration, the charcoal was washed with

water and 0.05 M HC1, and then eluted with acidic aqueous_ acetone. The resulting filtrate

was concentrated to a small volume and precipitated in acetone. After standing at 4 °C

overnight, the acetone was decanted and the remaining brown oily residue was dissolved

in 6 M HC1, precipitated in methanol, filtered and dried to afford the crude product.

The isolation of IA and 1B was carried out chromatographically on Amber lite CG-

50 by using a gradient (0.4-0.8 M) of ammonium acetate (pH 9.0) monitored by a dual

wavelength (254 and 280 nm) UV detector. A typical chromatogram is shown in Figure

12. Desalting was required next, and this step was first tested by using Sephadex G-10

17

column chromatography followed by recrystallization, but recrystallization gave poor

yields. The yield was later improved dramatically by simply desalting the mixture on a

DEAE-Sepharose CL-6B column (C1- form).

Synthesis

160

140 -

120 -

100 -

80

60

40

20 -

0

Unknown

ABS

Conc. (M)

IB

0

Fraction Number

Figure 12. Typical Chromatogram of Capreomycins

0.9

0.8

- 0.7

0- 0.6 0

80

0.5

0.4

DL- [2,3,3- 2H3]Dap (11a)

DL-Aspartic acid was used as the starting material to prepare DL-[2,3,3-2H3]Dap

(11a). Unlabeled aspartic acid was first converted to DL-[2,3,3-2H3]aspartic acid via an

18

exchange reaction in D20 catalyzed by pyridoxal and Al2(SO4)3.42 The resulting

product was then converted to DL- [2,3,3- 2H3]Dap by Schmidt reaction43 with fuming

H2SO4 and NaN3 (Scheme 5). The product contained deuterium at H-2 (98% enrichment)

and at H-3 (80% enrichment) as determined by 2H NMR spectroscopy.

Scheme 5

H H

11)

NH2

HOOC COOH

Fuming H2S041.NaN3

D20, NaOH

Al2(804)3, Pyridoxal

D DD NH2

H2N COOH

11a

HOOC

NH2

COOH

DL- [2- 2H]Dap (llb)

The first attempt to make DL-[2-2H}Dap (lib) via a cobalt complex was

unsuccessful. No DL-[2-2H]aspartic acid, which would have then been converted to lib

as described above, was obtained. A possible explanation is that deuteriated aspartic acid

could not be released from the aspartato-cobalt complex by chromatography. Both 1H

and 2H NMR spectra suggested the existence of the aspartato-cobalt complex. In the

second attempt, llb was prepared from DL-Dap by an acid-catalyzed exchange reaction

(Scheme 6).45 DL-Dap was mixed with D20 and 35% DC1, and heated at 125 °C in a

sealed pressure tube for 48 h. After removal of 1320 and DC1, the residue was redissolved

in a small volume of water and precipitated in absolute ethanol. The final product showed

a 97% deuterium enrichment at H-2 as determined by 2H NMR spectroscopy.

Scheme 6

H HH NH2

H2N COOH

D20, 35% DCI70-

H DH NH2

H2N COOH

19

Hydrolysis of Capreomycin and Purification of Capreomycidine (5)

The hydrolysis of capreomycin has been reported,1 and the capreomycidine (5)

thus obtained was purified by electrophoresis. In our work, crude capreomycins (1 g)

obtained by fermentation was hydrolyzed in 6 M HC1 at 125 °C for 48 h. The

hydrolysate was then passed through a Dowex 50W-X8 column and the column was

washed with water until the elutant pH was 6-7. Serine and alanine were removed during

this step. The column was then eluted with 0.3 M NH4OH, and ninhyrin-positive

fractions were combined and evaporated to a small volume. This residue was further

purified by chromatography on a Dowex 50W-X8 column using a gradient of

(NH4)2OAc buffer (pH 7-9) to give a 57% recovery of 5 based on a theoretical yield (160

mg, considering 60% of 1 and 2 in crude product) after desalting by DEAE-Sepharose

CL-6B chromatography.

The deuterium-hydrogen exchange reaction at the a-position of 5 was tried using

the same method as that of 11b, but it was unsuccessful. More than one position had

been labeled by 2H. Although it was not a desired product, the multiply deuteriated 5

could still be used as a precursor to test whether or not 5 is a free intermediate in the

biosynthesis of capreomycin.

20

Feeding Experiments

L-[U-14C]Serine, L-M-14C1Alanine and L-[U-14C]Cystine

Administration of L- [U- 14C]serine to fermentations of S. capreolus yielded a

2.1% incorporation for 1 and an 1.6% incorporation for 2, whereas administration of

either L- [U- 14C]alanine or L-[U-14C]cystine yielded less than 1% incorporation into

either 1 or 2. These results, shown in Table 1, indicated that serine is the most likely

precursor not only for the serine residue in 1, but also for the 2,3-diaminopropionate

(Dap, 11) residues of 1 and 2.

Table 1. Results of Radioactive Precursor Feedings

Precursor Specific Activity (dpm/mg)Based

% Incorporation

L-[U -14C1-serine IA 2.04 x 103 IA 2.05IB 1.58 x 103 IB 1.60

[U--[U-14C1-alanine IA5.29 x 102 IA 0.50

IB 9.29 x 102 IB 0.90

;-[U-14C]-cystine IA5.12 x 102 IA 0.46

IB 6.96 x 102 B3 0.631

DL-[1-13C]Serine (10a)

Cultures of S. capreolus were fed DL-[1-13C]serine (10a) to obtain positional

information on the origin of the residues that make up capreomycins. Figures 13a and

13b give the specific enrichments that were determined from the 13C NMR spectra of the

derived lb and 2b, normalized to the C-1 resonance of the natural abundance spectrum.

21

Enrichments (average 1.0%) at C-10, C-17 and C-34 clearly revealed that serine is a

primary precursor for the Dap residues and the .62,3-ureido-Dap (Uda) residue. The

dramatically higher enrichment at C-30 of lb compared to that of at other positions

confirmed that 10a was incorporated directly into the serine residue (Figure 13a). These

results suggest that the capreomycin pathway involves pre-peptide assembly modification

of serine to Dap, rather than post-peptide assembly modification of serine residues, since

approximately the same enrichment at all four carbonyl positions should result if serine

were incorporated directly into each. In contrast to lb, similar enrichments at all four

equivalent positions of 2b were observed. This result appeared to rule out the possibility

that capreomycin IA, 1, is derived by hydroxylation of capreomycin IB, 2. Minor

incorporation of the labeled serine into the guanido-group of the capreomycidine residue,

the ureido-group of the modified Dap residue and into the 13-lysine residue could be

rationalized by the metabolic pathways shown in Figure 14.

0.83%O

H2N ,,,,,3 4

H 1

30

1

0NH / HN 1 7 o

N 1 0 N

II I0) HC..1 NH

N NH2+

0.24%I

0.55%

0 NH2

N 21

1 NH2

0 LT:Zimi

NH2

Figure 13a. Enrichments over Natural Abundance Observed in lb

23

CH3-J%CO2-

Pyruvate

OH

H3N" CO2"

Serine

CO2

O 0

H2N CH2- CH2 - OH

Ethanolamine

CH3

H311. 6,02

Alanine

c02-

H3N' CO2

Aspartate

CHO

1I-13N* CO,-

Aspartyl Semialdehyde

H2N - 3: -

Carbamoyl Phosphate

1111

HN - NH2HN C: NH2

(CH2)3 CH

H3N* )1..' CO2" H3N* '11. 002-

Citrulline 0-U reido-dehydroalanine

H3N*. 2-

(C,

C0

(CH2)3

1-1314. "..% CO2-

Diaminopimelate

1NH2

(CH2)4

).H,N co,-a-Lysine

NH2

(CH2)3

H3N` CH

CH2.1CO2"

(3-Lysine

*NH2n

HN - C: NH2

(CH2)3

..1%.I-13N+ CO2

Arginine

-0 `NH3

NH

N oi *NH2H

Capreomycidine"

"c .13c

Figure 14. Proposed Metabolic Pathways for Incorporation of [l-13C1Serine into f3-

Lysine, Capreomycidine and 13-Ureido-dehydroalanine

24

DL-[3-13C]Alanine (12a)

DL-[3-13C]Alanine (12a) mixed with L- [U- 14C]alanine was fed next, and yielded

lc and 2c with the enrichments shown in Figures 15a and 15b. The 3.0% enrichment at

C-32 of 2c in comparison to significantly lower enrichments at other enriched sites was

consistent with the direct incorporation of 12a into 2c. No enrichment was observed at

Dap or Uda residues. Again, secondary incorporation was observed in the

capreomycidine, Uda and 13-lysine residues, and could be rationalized by the metabolic

pathways shown in Figure 16.

NH

NH H' HN

N NH2

NH2

Figure 15a. Enrichments over Natural Abundance Observed in is

25

NH

.0NH H° FIN 0

I HN N NH2

Figure 15b. Enrichments over Natural Abundance Observed in 2c

26

CH3

H3N+

Alanine

H2N- CH2

CH2

CH2.1

H3N+ CH

CH2

c02-(3-Lysine

?1-13

Off` CO2-

Pyruvate

CH3

SCoA

Acetyl CoA

"02C\CH3

d%* CO2

Oxaloacetate

TCA cycle

.02C% -02C% -02C%

CH2 CH2 CH21 1 1

CH2 CH2 CH2

041 0- OA SCoA 0 CO2"

Succinate Succinyl CoA a-Ketoglutarate

CH3 CH3

A ._0 uul 0#.' CO2

Pyruvate Pyruvate

pi

Gri +N H3

NH

N +NH2

Capreomycidine

,3c

CO2

2 2H2N-0:0-F:-

0_

Carbamoyl Phosphate

+NH2

HN-- C- NH2

CH2

CH2

CH2

143N+..°14 CO2

Arginine

02C%

oH2

CH2

H3N+-...k CO2

Glutamic Acid

NH2

CH2

CH2

CH2

H3N+'11. CO2

Omithine

Figure 16. Proposed Metabolic Pathway for [3-13C]Alanine Feeding

27

DL-[2,3,3-2H3]Serine (10b)

In order to determine the mechanism for conversion of serine to Dap, DL-[2,3,3-

2H3]serine (10b) was fed next and yielded ld and 2d. These were analyzed by 2H NMR

spectroscopy in 2H-depleted water, and FIDs were acquired at 298 K. Since the resonance

frequency for H-31 (84.84) overlapped with the small remaining solvent signal, a second

FID was acquired at 345 K with a pre-saturation pulse to diminish the residual water

resonance. The spectrum of 2d (Figure 17b) at 345 K clearly showed resonances for H-12

(88.07), H-19 (83.86 and 83.71), H-26 (81.82), H-32 (81.44) and H-37 (84.15 and

83.29). No deuterium was observed at H-18 (64.43) or H-35 (84.34). Retention of the (3-

hydrogens and loss of the a-hydrogen would be consistent with involvement of a

dehydroalanyl residue in the conversion to 11. The 2H NMR spectrum of ld (Figure 18b)

at 345 K was almost the same as that of 2d except for an additional resonance at H-31

(84.84), which again was consistent with direct incorporation of serine at serine residue in

IA and replacement of serine by alanine at the alanine residue in IB. The enrichment

appearing at H-26 (81.82) could also be rationalized by the metabolic pathway shown in

Figure 14.

b

a

H-19 HODH-32

28

f l 1 i 1 1 i i 1 1 1 T I -I -I 7 I

10 9 8 7 6 5 4 3 2 1 0PPM

Figure 17. NMR Spectra of 2d. (a). 400-MHz 1H NMR spectrum in D20 at 298 K. (b).

61.4-MHz 2H NMR spectrum in 2H-depleted H2O at 345 K showing deuterium

enrichment at H-12, H-19, H-26, H-32, and H-37; residual HOD signal (84.4) was

suppressed by inversion recovery water suppression pulse.

29

I I 1 1 I 1

10 9 8 7 6 5 4 3 2 1 0PPM

Figure 18. NMR Spectra of id. (a). 400-MHz 1H NMR spectrum in D20 at 298 K. (b).

61.4-MHz 2H NMR spectrum in 2H-depleted H2O at 345 K showing deuterium

enrichment at H-12, H-19, H-26, H-31, H-32 and H-37; residual HOD (M.4) was

suppressed by inversion recovery water suppression pulse.

30

DL-[2,3,3-2H3]Dap (11a)

Results from the serine feedings clearly indicated conversion of serine to 11 prior

to peptide assembly. To test this directly, DL- [2,3,3- 2H3]Dap was fed to S. capreolus, and

the derived metabolites, le and 2e, were analyzed by 2H NMR. Both spectra (Figure 19b

and Figure 20b) clearly show deuterium enrichment at H-12, H-19 and H-37. By

comparison with the spectra of id and 2d, deuterium enrichments at H-18 and H-35 were

also recognizable. No enrichment was observed for the serine or alanine residue of le or

2e, respectively. The approximate enrichments were determined by cutting and weighing

traces of the relevant signals from capreomycin and from the t-BuOH that had been

included as a chemical shift reference and for deuterium quantitation. This yielded an

enrichment of 25% at H-12, and an average of 14% for each of the other sites in 2e. The

values for le were 16% and 5%, respectively. 2,3-Diaminopropionic acid is clearly an

intermediate in the biosynthesis of capreomycin, and is thus formed prior to the formation

of the pentapeptide.

DL-[2-211]Dap (11b)

In order to more clearly define the fate of H-2 of 11a, DL- [2- 2H]Dap (11a) was

fed to S. capreolus. The 2H NMR spectra of if and 2f thus obtained (Figure 19c and

Figure 20c) exhibited the expected resonances (E4.3-84.5), but contained less deuterium

at H-18 and H-35 than expected from the previous feeding. However, we subsequently

determined that at these positions the capreomycins suffer slow exchange (ca. 25-50%,

see below) under the fermentation conditions. Absolute levels of apparent incorporation

were observed to vary between experiments more than would typically be observed just

from the vagaries of two different biological experiments.

b

aH-12

HOD

H-19'

H-37'

H-18

H-35

8 7

-6 5

PPM4 3

H-19

H-37

31

Figure 19. Partial NMR Spectra of le and lf. (a). 400-MHz 1H NMR spectrum of 1 in

D20 at 298 K. (b). 61.4-MHz 2H NMR spectrum of le in 2H-depleted H2O at 345 K

showing deuterium enrichment at H-12, H-19 and H-37; residual HOD (84.4) was

suppressed by inversion recovery water suppression pulse. (c). 61.4-MHz 2H NMR

spectrum of if in 2H-depleted H2O at 298 K showing deuterium enrichment at H-18 and

H-35.

b

a

9 8 7 6 5 4 3PPM

32

Figure 20. Partial NMR Spectra of 2e and 2f. (a). 400-MHz 1H NMR spectrum of 2 in

D20 at 298 K. (b). 61.4-MHz 2H NMR spectrum of 2e in 2H-depleted H2O at 345 K

showing deuterium enrichment at H-12, H-19 and H-37; residual HOD (54.4) was

suppressed by inversion recovery water suppression pulse. (c). 61.4-MHz 2H NMR

spectrum of 2f in 21I-depleted H2O at 298 K showing deuterium enrichment at H-18 and

H-35.

33

Exchange Reaction

Deuterium-hydrogen exchange under simulation of the fermentation conditions

was carried out by first using pure capreomycin IB, 2, in D20 at pH 8.0, 30 °C, 250 rpm

(in a shaker) for 7 days. The reaction mixture was then freeze-dried and desalted on a

DEAE-Sepharose CL-6B column. Both 1H and 2H NMR spectra of the sample showed a

similar (ca. 50%) deuterium enrichment each at 84.3-4.4 and 85.0, which correspond to

H-18 (and/or H-35) and H-2 in 2. Next, 36 mg of the resulting product was treated with

H2O and the above procedure was repeated. Approximately 20% of the deuterium was

exchanged back to protium. Another 20 mg of the initial enriched product was mixed

with unlabeled capreomycin IB to make a final deuterium enrichment of 5%,

approximately the expected level of enrichment in our feeding experiment. The sample

was dissolved in H2O and placed in a shaker under the same conditions for 4 days. The

product contained ca. 2% deuterium enrichment at these same positions. The latter result

was consistent with the former result in that ca. 50% deuterium-hydrogen exchange had

occurred under the fermentation conditions.

A summary of all of the feeding experiments is shown in Figure 21.

H2N

NH2

CO2H

11, H2 =H3 =H OH11a, H-2 =H-3 =D11b, H-2= D, H-3= H

H2N CO2H CH3

10a, C-1 =13C H2N)%4CO2H10b, H-2 = H-3 = D

HO2C NH2

NH2

N/L NH

8, H-2 = H-3 = H-5 = H8a, H-2 = H-3 = H-5 = D

H2N3/4,NH

NH

12a, C-3 = 130

H0 NH2

33 NN 21 23 25

0 HHN ° -111H

NH0

5N NHH

la, R=OH, H-3= H-5 =Dlb, R=OH, C-10 = C-17= C-30 =C-34 =13Clc, R =OHld, R=OH, H-12 = H-19= H-31 =H-32 =H-37 =Dle, R =OH, H-12= H-19= H-37 =Dlf, R=OH,H-18 =H-35 =D

2a, R=1-1, H-3 =H-5 =D

2b, R H, C-10 = 0-17 = C-30 = C-34 .13D2c, R = H, C-32 =13C

2d, R = H-12 = H-19 = H-37 = D

2e, R=H,H-12=H-19=H-37=D2f, R=H, H-18 =H-35 =D

34

Figure 21. Summary of Labeling Patterns of 1 and 2 From Feeding Experiments with

Precursor Amino Acids

35

Studies with Cell-free Extract from. S. capreolus

From feeding various labeled precursors to S. capreolus, it was clear that serine

was converted into Dap before a cyclic pentapeptide was formed, and arginine was

possibly incorporated into a linear peptide prior to the formation of capreomycidine.18

Serine and alanine also were directly incorporated into 1 and 2, respectively. Although

the mechanism of the peptide assembly leading to capreomycin was still unknown, based

on the results from studies of other peptide biosynthesis,48-51 it is reasonable to assume

that all the precursor amino acids were activated directly at their respective enzyme sites

before the formation of the cyclic pentapeptide. Therefore, studies at the cell-free level

were required in order to better understand the details of the biosynthesis of capreomycin.

Generally, compounds containing non-protein constituents in the polypeptide

chain may be derived from either modification of the chain or originate from non-

ribosomal systems,40 whereas protein antibiotics (containing only protein amino

acids)46,47 are usually formed via ribosomal mechanisms. In non-ribosomal systems, the

activation of constituent amino acids usually occurs as a result of either ATP

[32P]phosphate exchange,48 corresponding to a "non-thiotemplate" mechanism, or ATP -

[32P]pyrophosphate exchange, corresponding to a "thiotemplate" mechanism as observed

in other peptide antibiotics (Scheme 7),49-51 leading to the formation of an enzyme-

stabilized, non-covalently bound amino or hydroxyl acyladenylate. Upon addition of

[32P]pyrophosphate, the reaction is reversed and [32P]ATP is formed. Assays for

detecting the enzyme activity are usually carried out by using this protocol.

36

Scheme 7

EnzymeATP + substrate substrate-AMP + PP;

Thiotemplate mechanism

Or

EnzymeATP + substrate substrate-ADP + Pi

Non-thiotemplate mechanism

Preparation and Partial Purification of Cell-free Extract (CFE)

Initially, mycelia of S. capreolus were collected from a 5-day fermentation culture

on the basis of capreomycin accumulation (since this corresponded to maximal

capreomycin production). In similar cases involving activating enzymes from other

Streptomyces strains, protein from younger cells was used for enzymatic studies,50 -52

probably due to a higher concentration of enzyme at early stage of cell growth. In our

studies higher enzyme activity was detected when using S. capreolus cells harvested 48 h

after inoculation. Cells were collected by centrifugation, washed sequentially with

buffer, 1 M KC1, 0.8 M NaCl, and buffer. The cells could be stored at -80 0C for at least

two weeks after this step. Typically, one volume of mycelia was then resuspended in two

volumes of pre-cooled buffer, to which protease inhibitor phenylmethanesulfonyl fluoride

(PMSF) had been added, and disrupted by sonication. The CFE was obtained by

centrifugation. Polyethylenimine (PEI) cellulose was then added in order to remove

nucleic acids.53 In early experiments, the final concentration of PEI-cellulose was

usually brought to 0.3%. Subsequently, it was found that the amino acid activating

enzyme activity was increased if the final concentration of PEI-cellulose was reduced to

0.05% 0.1%. The precipitate from the PEI-cellulose procedure was removed by

37

centrifugation, and the resulting supernatant was treated with solid ammonium sulfate. In

one experiment, the solution was brought to 30% (no precipitation occurred at this step),

50% and 90% saturation with solid ammonium sulfate, respectively. In another

experiment, it was brought to 45%, 60% and 80% saturation. The pellets were collected

by centrifugation and desalted on PD-10 (Sephadex G-25) spin columns prior to

enzymatic assays.

Protein Concentration Determination

Protein concentrations were determined by the method of Bradford,54 using

bovine serum albumin (BSA) as the calibration standard. A standard calibration curve is

shown in Figure 22.

0 5

BSA (gg/mL)

10

Figure 22. Bradford Assay: BSA Standards

15

38

Based on the plot shown above, the protein concentration in the preparation of

enzyme for enzyme activity assay was calculated as shown below:

3.38 mg/mL protein in crude CFE.

2.79 mg/mL protein in the supernatant after PEI-cellulose precipitation, 32% of

total protein was removed in this step.

2.19 mg/mL protein in the supernatant of 0-45% (NH4)2SO4 precipitation, 16% of

total protein was removed by (NH4)2SO4.

1.38 mg/mL protein in the supernatant of 46-60% (NH4)2SO4 precipitation, 20%

more of total protein was removed by this step.

Only 0.27 mg/mL protein remained in the supernatant of 61-80% (NH4)2SO4

precipitation. 26% of total proteins were in the pellet.

The protein concentrations for the enzyme assays were as follows:

2.72 mg/mL in 0-45% (NH4)2SO4 pellet.



Enzyme Assay

Amino acid dependent ATP/phosphate and ATP/pyrophosphate exchange

reactions were carried out by a modification of the method of Lee and Lipmann.55 Assay

conditions for enzymatic synthesis of [32PJATP from either [32P]phosphate or

[32P]pyrophosphate were similar to those of Keller.49-51 In a total volume of 500 pL,

250 pL of enzyme, 10 mM phosphate buffer (pH 7.5), 2.5 mM ATP, 4.0 mM MgC12, 10

mM each of substrates, and 50 p.1_, [32P]phosphate or [32P]pyrophosphate (5 x 105 dpm)

were incubated for 30 min at 32 °C. The reaction was stopped by the addition of 10%

trifluoroacetic acid, and radioactive ATP was separated from [32P]phosphate or

[32P]pyrophosphate by adsorbing it onto activated charcoal. [32P]Phosphate and

[32P]pyrophosphate do not bind to charcoal and could be washed out with water, whereas

39

[32P]ATP remained bound to charcoal under these conditions. Radioactive ATP was

eluted from charcoal with either 6 M HC1 or 10% aqueous pyridine and then counted by

liquid scintillation counter. In several control experiments that have been performed in

order to find the ideal assay conditions, 10% aqueous pyridine demonstrated a higher

efficiency in removal of radioactivity from charcoal.

The details of a typical set of assays are shown below:

Vial # Substrate Enzyme

1 Dap Fresh

2 Arginine Fresh

3 Alanine Fresh

4 Serine Fresh

5 Aspartic Acid Fresh

6 Water Fresh

7 Dap Boiled (2 min)

8 Dap No Enzyme

Several early attempts to find the enzyme activity were hampered by a high

background exchange rate. This problem was later resolved by changing the brand of

charcoal (that purchased from either Spectrum Chemical Mfg. Corp. or Eastman showed

the higher background than that of Alpha) and decreasing the amount of charcoal used for

assays. The results from ATP /[32P]phosphate exchange assays with crude CFE suggested

that no exchange had occurred. All of the assays gave the same level of the radioactivity

(a few hundred dpm above the background) after ATP was eluted from charcoal, no

matter which substrate was used. The results from ATP/[32P]pyrophosphate exchange

40

with a variety of enzyme preparations indicated the presence of the amino acid activating

enzyme(s). The highest specific activity was found in the fraction of 0-45% (NH4)2SO4

saturation.

The results from one set of assays are listed in Table 2.

Table 2 . Specific Activity of t2P]ATP from ATP/[321113Pi Exchange Reaction.

(initial 2111)13i: 5.15 x 105 dpm)

l'Op,".12-, substrate

ay472,

/l

Enzymes

Dap Arg Ala Ser

Asp

(Non-precursorsubstrate)

NoSubstrate

(Endo-genous)

Dap

BoiledEnzyme

Dap

NOenzyme

Crude CFE 17515 22189 25089 25965 15858 32024 234 494

0-45% (NH4)2SO4

saturation pellet . 33206 31176 57029 50118 14235 25059 324 370

46-60% (Nli)2SO4

saturation pellet 6460 6418 7372 7565 7180 4753 510 636

61-80% (Nli)2SO4

saturation pellet 3729 3627 4424 3188 4812 4973 406 580

Apparently, the amino acid activating enzyme(s) precipitated at a concentration of

0-45% ammonium sulfate saturation and all of the precursor amino acids were activated

by the enzyme(s). The endogenous exchange rate still remained high in these assays with

the reasons unknown. However, it seemed to decrease after several purification steps. The

non-constituent amino acid of capreomycin, aspartic acid, was also activated in all the

assays performed. This might be accounted for that we chose improper substrate as one of

the controls. It is possible that the enzyme(s) may have a specific site that can activate

aspartic acid which is not utilized in the biosynthesis of capreomycin.

In order to demonstrate that the recovered 32P was associated with ATP produced

in assays with the precursor amino acids, but not with other controls, an autoradiographic

41

study was carried out. An autoradiogram (sensitive to as low as 1000 dpm) of a PEI-

cellulose TLC chromatogram of various exchange reaction mixtures containing different

substrates and enzymes showed the presence of [32P]ATP in assay mixtures with all the

amino acids as substrate, but not with boiled enzyme or incubations lacking enzyme. In

addition, the assay mixtures with the precipitate of 0-45% (NH4)2SO4 saturation showed

the strongest signals on the TLC plate. This was consistent with the results observed from

LSC shown in Table 2. TLC with commercially available [32P]ATP (from NEN) which

was used as a reference showed two spots. One of them had the same Rf value as that of

[32P]pyrophosphate (from NEN). This may be due to degradation of [32P]ATP to AMP

and [32P]pyrophosphate.

Conclusions and Areas for Further Study

We have established that one equivalent of serine and alanine is incorporated

directly into 1 and 2, respectively. However, three of the remaining residues of each core

cyclic pentapeptide are indirectly derived from serine via a 2,3-diaminopropionic acid

intermediate (11). The apparent 0-elimination/replacement mechanism revealed by

incorporation of the deuteriated amino acids is consistent with previous studies on the

biosynthesis of malonomicin56 in particular, and on pyridoxal phosphate-dependent (3-

replacement reactions57 in general. The clean incorporation of added 11 into the Dap and

Uda residues of 1 and 2 is consistent with a pre-peptide assembly modification of serine,

rather than a post-peptide assembly modification (e.g. simple dehydration) such as

presumably results in the dehydro-alanyl residues of berninamycin58 and nosiheptide.59

In contrast with the incorporation of 11, results from the previous study18 of

arginine incorporation are consistent with post-peptide-assembly modification to yield an

N-acyl-dehydroarginyl residue which then cyclizes. The fl-ureido-dehydroalanine residue

most reasonably also comes from post-peptide assembly modifications of a peptide Dap

42

residue. Figure 23 shows two possible peptide assembly modifications which need to be

further investigated in order to get a clearer picture of this biosynthetic process.

H2N Dap-Ala-Dap- Dap-A rgCOOH

H2NDap-Ala-Dap-Dap-CpdCOOH

H2NDap-Ser-Dap-Dap-ArgCOOH

H2N Dap-Ser-Dap-Dap-CpdCOOH

Dap-Ala-Dap-Dap-Cpd

NHCO

Capreomycin IB

Hydroxylation

Dap-Ser-Dap-Dap-Cpd

NHCO-1Capreomycin IA

Figure 23. The Apparent Manner of Capreomycin Assembly

The enzyme activity assay results revealed that the amino acid activating enzyme

activities for serine, arginine, alanine and Dap were present in the soluble supernatant of

the capreomycin producer cells, and that the biosynthesis of capreomycin appears to

involve activation of the component amino acids to produce substrate-AMP, as expected

for a non-ribosomal "thiotemplate" mechanism.40 This result illustrates another example

of the multi-functional/multi-enzyme complexes involved in the biosynthesis of microbial

peptide antibiotics. With some exceptions, most of these appear to involve a pantotheine

arm, presumably for covalent attachment of a growing peptide. Although a number of the

peptides studied have had an unusual amino acid starter unit, the succeeding residues

43

have generally been either intact protein amino acids or ones that have undergone

relatively simple modifications such as a-epimerization or N-methylation within the

peptide synthetase complex.38

The capreomycins present an opportunity to study the mechanisms and

relationships of extensive pre- and post-peptide assembly modification, in addition to the

peptide assembly process itself. Further characterizations of the enzyme(s) are clearly of

interest.

44

Experimental

General

S. capreolus A250 was obtained from the Eli Lilly Company. Bacillus subtilis

ATCC 6633 was obtained from Becton Dickinson Microbiology Systems. Carbon nuclear

magnetic resonance spectra were recorded at 75.5 MHz on a Bruker AC 300 (300 MHz

for 1H) spectrometer, and proton decoupled 2H NMR spectra were run unlocked at 61.4

MHz on a Bruker AM 400 (400 MHz for 1H) spectrometer. All NMR spectra were

obtained using 5 mm NMR tubes, and all samples were prepared in either D20 or 2H-

depleted H2O. The chemical shifts are reported in parts per million (ppm) relative to an

internal standard of t-BuOH (81H=1.28, 82H=1.28, 613C= 31.2).

Radioactivity measurements were carried out using a Beckman Model LS 7800

Liquid Scintillation Counter (LSC) with automatic quench correction and external

standardization to yield disintegrations per minute (dpm).

Amber lite CG-50 ion exchange chromatography was carried out at 4 °C. DEAE-

Sepharose CL-6B column chromatography was performed at room temperature.

Activated charcoal used for work-up of capreomycins was purchased from Spectrum

Chemical Mfg. Corp. Ion exchange resins were purchased from either Bio-Rad

Laboratories or Sigma Chemical Company.

Cell disruption was carried out by using a sonicator (Model W-225R, Heat

System-Ultrasonics, Inc.). Activated charcoal for enzyme activity assays was purchased

from Alpha Products (Johnson-Matthey). Protein assay kit was purchased from Bio-Rad

Laboratories. UV measurements were performed on an IBM 9420 UV-Visible

spectrophotometer.

L- [U- 14C]Serine, L- [U- 14C]alanine and L-[U-14C]cystine were purchased from

ICN. DL- [1- 13C]Serine and DL-[3-13C]alanine were purchased from Cambridge Isotope

45

Laboratories. Deuterium depleted water for 2H NMR was purchased from Aldrich

Chemical Company.

Fermentation of S. capreolus A250

Maintenance of S. capreolus A250

The agar medium (200 mL) contained dextrin (Difco, 2.0 g), yeast extract (Difco,

0.2 g), N-Z amine A (ICN Biochemicals, 0.4 g), beef extract (Difco, 0.2 g), CoC12.6H20

(0.002 g), BBL granulated agar (Bacton Dickinson Microbiology Systems, 4.0 g), and

deionized water. The pH was adjusted to 7.0 with 1 N NaOH prior to sterilization. Agar

in a petri plate was streaked with S. capreolus A250 spore suspension that had been

stored at -80 °C, and was then incubated at 28 °C for 7 days. An isolated colony was

suspended in 0.9% sterile NaC1, and a portion of the suspension (0.25 mL) was used to

inoculate another agar plate for confluent growth. After incubation at 28 °C for 27 days, a

sterile solution of 0.1% Tween-80 (10 mL) was added and the spores were suspended

with the aid of a sterile loop. The spore suspension was then transferred to a sterile

centrifuge bottle and centrifuged at 3000 rpm for 10 min. The pellet was resuspended in

20% sterile glycerol (12 mL) and 1.2 mL aliquots were distributed to cryovials and

preserved in an ultra-freezer (-80 °C) for long-term storage.

Production of Capreomycins

A portion of a spore suspension of S. capreolust8 (0.25 mL) was used to inoculate

a seed medium (60 mL) containing tryptone (Difco, 0.30 g), yeast extract (0.15 g),

glucose (0.90 g), MgSO4 -7H20 (0.18 g), FeSO4-7H20 (0.06 mg), CaC12.2H20 (0.015 g)

and deionized water. The pH was adjusted to 7.5 with 2% KOH before sterilization.

The flask (250 mL) was incubated at 30 °C, 250 rpm for 42 h, and a portion of the

seed culture (5% v/v) was used to inoculate production medium (200 mL in 1 L

46

Erlenmeyer flask) containing glucose (5.60 g), Bactopeptone (Difco, 3.00 g), N-Z amine

A (0.80 g), blackstrap molasses (purchased from local food co-op, 2.00 g), potato starch

(Sigma, 2.00 g), MgSO4.7H20 (1.00 g), CaCO3 (0.40 g), and hot tap water. The pH (6.5)

was unadjusted. The fermentation (200 mL) was incubated at 30 °C, 250 rpm for 6 days.

Isolation of Capreomycins IA and IB

The fermentation broth (200 mL) was harvested after 6 days of incubation by

filtration through Celite. The filter cake was washed with water (100 mL), and activated

charcoal (9.0 g) was added to the combined filtrate and washings. The mixture was

allowed to stand for 40 min at room temperature. The charcoal was filtered through

freshly prepared Celite and the filtrate was discarded. Successive washings of the filter

cake with water (60 mL) and 0.05 M HC1 (60 mL) were also discarded. The filter cake

was next eluted with acidic aqueous acetone (20% acetone, 80% 0.05 M HC1, v/v, 250

mL) in a period of 15 min. The acetone eluate was then concentrated in vacuo to a small

volume (ca. 5 mL) and transferred, with stirring, to acetone (80 mL) and the cloudy

mixture allowed to stand at 4 0C overnight. The light brown solution was decanted, and

the resulting reddish-brown oily residue was dissolved in 6 M HC1 (3 mL). The solution

was filtered into stirred Me0H (36 mL), and the precipitate was then collected by

filtration.

A column of Amber lite CG-50 (100-200 mesh, N'H4+ form) (1.0 x 42.5 cm) was

equilibrated in 0.4 M NH4OAc, buffered with NH4OH to pH 9.0 at 4 0C. The sample

(450 mg) was loaded onto the column in a small volume (3 mL) of the same buffer. The

column was eluted first with a gradient from 0.4 to 0.8 M NH40Ac (225 mL each in Bio-

Rad gradient former) and then isocratically with 0.8 M NH40Ac (300 mL). Fractions (8

mL each) were collected at 60 -min intervals and were monitored by an UV detector (254

and 280 nm) (Pharmacia). On the basis of the UV profile, fractions containing either IA

47

or IB were combined and freeze-dried. The acetate salt was chromatographed on a

column of DEAE Sepharose CL-6B (Sigma, 1.0 x 30 cm, form) eluted with water.

Bioassay

Sterile PA-7 agar (1.5% Bacto-agar and 0.5% Bacto-tryptone) was thawed and

then equilibrated at 55 °C in a water bath. Agar (20 mL) was dispensed into a petri plate,

allowed to solidify and another 6 mL of the same agar containing a diluted B. subtilis

ATCC 6633 saline suspension (0.027% final concentration) was then added and used for

the bioassay.

Sterile stainless steel wells (i.d. 5 mm, up to 6 per plate) were placed evenly on an

agar plate prepared as above, and the solution (100 [tL) to be assayed was placed in each

well. The plate was incubated at 37 °C for 24 h. The diameter of the inhibition zone was

then measured and used to calculate the concentration of the antibiotic from the standard

curve.

Synthesis

DL42,3,3-2H3]Dap (11a)

DL-Aspartic acid (2.66 g, 20 mmol), NaOH (1.60 g, 40 mmol), pyridoxal

hydrochloride (0.41 g, 2 mmol) and Al2(SO4)3.18H20 (0.33 g, 0.5 mmol) were washed

into a 50-mL flask with D20 (10 mL). The sample was vortexed vigorously and

lyophilized. Triethylamine (1.4 mL, 10 mmol) and D20 (20 mL) were added to the

sample, and the mixture heated at 50 °C for two days in an oil bath covered with

aluminum foil to minimize photodecomposition of the pyridoxal. The mixture was cooled

and the contents were washed into a 500-mL flask containing water (400 mL), sodium

oxalate (0.54 g, 4 mmol), 3-methylpyrazole (0.8 mL,10 mmol) and 1 M HC1 (35 mL, 35

48

mmol). The resultant mixture was then loaded onto a Dowex 50W-X8 column (H+ form,

100-200 mesh, 2.5 x 24 cm). After washing the column with water, the sample was

eluted with 0.1 M NH4OH. Ninhydrin positive fractions were combined, rotary

evaporated, redissolved in water, and re-evaporated to dryness. The residue was dissolved

in 25% DC1 (16 mL) and added to a flask which was then evacuated and sealed. After

heating for 8 days at 125 °C, the mixture was cooled, diluted with water, and evaporated

to dryness in vacuo . The sample was redissolved in water and taken to dryness to remove

excess HC1, yielding 0.80 g (30%) DL-[2,3,3-2H3] aspartic acid. mp: 301 °C dec. (lit-6°

>300 °C dec.); Integration of the peak at 8 4.09 and 8 2.95 from 2H NMR spectrum

showed the deuterations at H-2 (98%) and H-3 (97%); 1H NMR spectrum which was

obtained by using the same amount of sample used for 2H NMR study showed no

resonance at the corresponding chemical shift.

DL-[2,3,3-2H3]Aspartic acid (0.77 g, 5.66 mmol) was placed in a 250-mL 3-neck

flask and dissolved in 20% fuming sulfuric acid (5 mL) with cooling followed by the

addition of dry chloroform (15 mL). The mixture was heated to reflux, and sodium azide

(0.80 g, 12.32 mmol) was added in small amounts with vigorous stirring over 3 h. The

mixture was stirred for an additional 4 h, then cooled in an ice bath, and the chloroform

layer was poured off. The thick paste-like residue was poured onto crushed ice (ca. 30 g)

and diluted to 100 mL. The solution was then passed through a Dowex 50W-X8 column

(H+ form, 100-200 mesh, 2.5 x 11 cm) and washed first with 150 mL 0.5 M HC1, and

then with water to neutrality. The adsorbed lla was eluted with 10% ethanolic ammonia

(4.0 M), and the ninhydrin positive fractions were combined and evaporated to a small

volume (ca. 2 mL). Ethanol (95%, 20 mL) was added slowly and most of the llaHC1

precipitated. The precipitate was filtered, washed with 95% ethanol (10 mL) and dried.

The filtrate was concentrated to ca. 5 mL, and more 11a-HC1 was recovered. The

combined yield was 0.41 g (69%) as a white powder. mp: 234 °C (lit.61 236-237 °C);

49

2H NMR analysis indicated 98% deuteriation at H-2 and 80% at H-3. 1H NMR spectrum

showed only a very small peak at 8 3.5.

DL- [2- 2H]Dap (lib)

A solution of DL-diaminopropionic acid monohydrochloride (2.00 g, 19 mmol),

D20 (20 mL), and DC1 (35% in D20, 1 mL) was placed in a pressure tube, sealed and

heated in an oil bath at 125 0C for 24 h. After cooling, the solvent was removed in vacuo

and the resulting residue was dried under vacuum for 2 h. The residue was dissolved in

water (50 mL), concentrated to a small volume (ca. 3 mL), and precipitated in absolute

ethanol (100 mL). After standing at 4 0C for 4 h, the precipitate was collected by

filtration, washed with ethanol, and dried under vacuum to give 1.94 g (96%) of DL-[2-

21-11-Dap-HC1. mp: 234 0C; 1H NMR (D20, 300 MHz) 8 4.07 (t, < 0.03H, J=7.06 Hz), 8

3.50 (s, 2H); 2H NMR analysis indicated 97% deuteration at H-2.

Hydrolysis of Capreomycin and Purification of Capreomycidine (5)

Capreomycin (1.0 g, crude product from fermentation) was refluxed at 125 0C in

20 mL of 6 M HC1 over 48 h. The solvent was removed by evaporation under vacuum,

and the residue treated with water (100 mL) and again evaporated to dryness. The residue

was then taken up in 75 mL of water and passed over a Dowex 50W-X8 column (200-

400 mesh, H+ form, 2.5 x 12 cm).

The column was eluted with water until pH 6-7, and then eluted with 0.3 M

NH4OH until no ninhydrin-positive materials came off the column. All of the ninhydrin

positive fractions were combined and concentrated. The residue was then applied to a

Dowex 50W-X8 column (200-400 mesh, H+ form, 2.5 x 12 cm) and eluted with a pH

gradient (pH 7-9) of 0.4 M NH4OAc. Elution progress was followed by silica gel TLC

using the system CHC13 / Me0H / NH4OH / H2O (1:4:2:1). Fractions 5-20 (8-10 mL per

50

fraction) contained Dap and 21-25 contained a mixture of Dap and capreomycidine.

Fractions 26-30 contained capreomycidine exclusively while fractions 38-48 contained

another ninhydrin-positive material which was not identified.

Fractions 26-30 were combined and freeze-dried to give 110 mg of residue which

was then charged to a DEAE-Sepharose CL-6B column (2.5 x 15 cm, form). Elution

was carried out using water. Fraction volumes were 7-8 mL and the ninhydrin-positive

material appeared in fractions 3-9. These fractions were combined and freeze-dried to

yield 91.2 mg (57% based on theoretical yield) of pure capreomycidine. TLC: (CHC13-

Me0H-28%NH4OH-H20, 1:4:2:1, on silica gel, 0.25% ethanolic ninhydrin spray) Rf

0.46; 1H NMR (D20, 300 MHz) 8 4.13 (m, 1H), 8 3.95 (d, 1H, J=5.3 Hz), 8 3.47 (m,

2H), 8 2.20 (m, 1H), 6 2.02 (m, 1H); 13C NMR (D20, 75.5 MHz) 8 172.45, 155.91,

58.10, 50.23, 37.75, 23.43.

Feedings

Feeding Protocol

Labeled compounds, except DL- [2- 2H]Dap, were pulse-fed in thirds to production

cultures at 12, 32 and 56 h after inoculation with a seed culture. DL-[2-2H]Dap was fed in

four pulses at 12, 32, 56 and 80 h after inoculation.

Generally, a small volume (2-3 gL) of a stock solution of radioactive compound

was diluted with deionized water in a 10 mL volumetric flask. Duplicate samples of 50

IL each were diluted to 5 mL in volumetric flasks. Duplicate samples of 100 gL each

were taken for scintillation counting and the remainder was used for the feeding. The

stable isotopically labeled compounds were weighed out, and dissolved in 10 mL water

for the feeding. Each precursor solution was slowly introduced into the production broth

at the appropriate time using a syringe fitted with a MSI membrane filter (size 0.22 gm).

51

Calculations in Feeding Experiments

Various calculations were performed to determine the % incorporation, %

enrichment and specific activity based on the amount of precursor fed, bioassay and

radioactivity data. The formulae used for these calculations are given below:

Specific activity isolated =radioactivity (dpm) in metabolite isolated

mmole of metabolite isolated

specific activity isolated x mmole of metabolite produced*% Incorporation = x 100

total radioactivity (dpm) in precursor fed

* based on bioassay

gmole of 211 observed by NMR integration% 2H Enrichment = x 100

gmole of product used for 2H NMR

% 13C Enrichment =II Jr

)1r If

where Il = The absolute intensity of the signal to be measured in thelabeled sample

= The absolute intensity of the same signal (same chemicalshift) as above in the natural abundance sample

Ir = The absolute intensity of the reference signal which should notbe enriched in the labeled sample

= The absolute intensity of the same reference signal in thenatural abundance sample

L-[U-14C]Serine, L- [U- 14C]Alanine and LtU-14C]Cystine

A single seed culture was used to inoculate three production broths. Aqueous

solutions (10 mL each) of L- [U- 14Clserine (10.73 pCi), L- [U- 14C]alanine (11.02 pCi)

and L- [U- 14C]cystine (11.98 pCi) were each fed in three equal portions to separate 200 -

mL production cultures at 12, 32, and 56 h after inoculation. After a total of 6 days,

52

bioassay of the fermentations indicated ca. 480 mg of total capreomycins for each culture.

Work-up and purification yielded 113.5 mg of IA (specific activity 1.36 x 106

dpm/mmol, 2.1% incorporation based on one-half of the bioassay mass) and 177.0 mg of

lB (1.56 x 106 dpm/mmol, 1.6% incorporation based on one-half of the bioassay mass)

from the L- [U- 14C]serine feeding. Similarly, 78.5 mg of IA (3.54 x 105 dpm/mmol, 0.5%

incorporation) and 70.8 mg of 1B (6.07 x 105 dpm/mmol, 0.9% incorporation) from the L-

[U -14C]alanine feeding, and 133.7 mg of IA (3.42 x 105 dpm/mmol, 0.5% incorporation)

and 107.2 mg of IB (4.54 x 105 dpm/mmol, 0.6% incorporation) from the L-[U-

14c] cystine feeding were obtained.

DL- [l- 13C]Serine (10a)

DL- [1- 13C]Serine (10a, 350 mg, 3.30 mmol) was fed in thirds to a 200-mL

production culture at 12, 32, and 56 h after inoculation with a seed culture. Bioassay

indicated 490 mg total capreomycins. This was worked up as usual, and 340 mg of crude

product was obtained after methanol precipitation. Only the center fractions from each

peak obtained from the Amberlite CG-50 chromatography were combined, and pure lb

(79.9 mg) and 2b (45.4 mg) were collected after the final desalting with the DEAE

Sepharose CL-6B column. A portion of each (40.0 mg) and t-BuOH (25 ilL) were

dissolved in D20 (0.5 mL), and the 13C NMR spectra (75.5 MHz) obtained with sweep

width 18,518 Hz, 64 K data points, 300 pulse width, 1.769 sec aquisition time, and

36,378 scans (lb) or 25,600 scans (2b). Each FID was transformed with 3.0 Hz line

broadening.

DL-[3-13CJAlanine (12a)

A mixture of DL-[3-13C]alanine (12a, 445 mg, 4.94 mmol) and L- [U- 14C]alanine

(9.52 liCi) in water (10 mL) was pulse-fed to a 200-mL production culture as described

above. After work-up and purification, the center fractions yielded 74.7 mg pure is (1.59

53

x 105 dpm/mmol, 0.3% incorporation) and 118.5 mg pure 2c (3.35 x 105 dpm/mmol,

0.7% incorporation). The 13C NMR spectra were acquired with 22,050 scans for lc and

18,450 scans for 2c.

DL-[2,3,3-2H3]Serine (10b)

A mixture of D L-[2,3,3-2H3]serine (10b, 440 mg, 4.15 mmol) and L-[U-

14C]serine (8.06 pCi) in water (10 mL) were pulse-fed to a 200-mL production broth as

described above. After work-up and purification, 152.2 mg pure 1 d (2.63 x 105

dpm/mmol, 0.8% incorporation) and 135.3 mg pure 2d (4.57 x 105 dpm/mmol, 1.5%

incorporation) were obtained. Samples of each (20.0 mg ld and 31.7 mg 2d) in

deuterium-depleted water (0.5 mL) plus t-BuOH (25 !IL) were analyzed by 2H NMR

(61.4 MHz) at 298 K and 345 K, as previously described.18 36,860 scans at 298 K and

14,679 scans at 345 K were acquired for ld, and 54,180 scans at 298 K and 12,288 scans

at 345 K for 2d.

DL-[2,3,3-2H3]Dap (11a)

DL-[2,3,3-2H3]Diaminopropionic acid (11a, 410 mg, 3.94 mmol) in water (10

mL) was pulse-fed to a 200-mL production broth as described above. After work-up and

purification, 47.3 mg pure le and 32.6 mg pure 2e were obtained. Each sample in

deuterium-depleted water (0.5 mL) plus t-BuOH (25 "IL) was analyzed by 2H NMR (61.4

MHz) at 298 K and 330 K, is previously described.18 1,892 scans at 298 K and 11,264

scans at 330 K were acquired for le, and 2,183 scans at 298 K and 13,312 scans at 330 K

for 2e.

DL-[2-211]Dap (11b)

DL- [2- 2H]Diaminopropionic acid (11b, 800 mg, 7.70 mmol in 30 mL water, pH

was adjusted to 7 with Na2CO3 prior to feeding) was fed in four equal portions to a 200-

54

mL production broth as described above. The work-up was carried out without the

methanol precipitation step. After purification with Amber lite CG-50 column (NH4+

form, 100-200 mesh, 1.0 x 42.5 cm), 62.3 mg if and 54.0 mg 2f were obtained. Desalting

step was omitted in this experiment. Samples of each (26.6 mg if and 54.0 mg 2f) in

deuterium-depleted water (0.5 mL) plus t-BuOH (50 pl.) were analyzed by 2FI NMR

(61.4 MHz) at 298 K; 36,115 scans were acquired for lf, and 27,500 scans for 2f.

Exchange Reaction of Capreomycin 11B under the Fermentation Conditions

Pure unlabeled capreomycin B3 (100 mg) was dissolved in 10 mL of D20 and

adjusted pH to 8.0 by addition of sodium carbonate. The mixture was then placed in a

shaker and incubated for 7 days at 30 °C, 250 rpm. After removal of the solvent, the

residues were redissolved in a small volume of water and passed through a DEAE-

Sepharose CL-6B column (2.5 x 15 cm, Cl- form), eluting with water. The ninhydrin-

positive fractions were combined and freeze-dried to give 92 mg of product. A portion of

this product (17.7 mg) was used for NMR studies. Both 1H and 2H NMR analysis showed

ca. 46% deuterium enrichment at 84.3-4.4 (H-18 or H-35) and 85.0 (H-2), respectively.

Another portion of the deuteriated product (36 mg) was treated with H2O by the

same procedure as described above to see if any deuterium could be exchanged with the

protons in the solvent. After 4 days, ca. 28% deuterium enrichment remained. This

suggested ca. 40% exchange. In the third experiment, the deuteriated product (20 mg)

was mixed with unlabeled capreomycin IB (180 mg), dissolved in 20 mL of H2O and

adjusted pH to 8.0 with sodium carbonate. 2H NMR at this stage showed ca. 5%

deuterium enrichment at the corresponding positions. The mixture was then shaken under

the same conditions as described above for 4 days. After work-up and desalting steps, 70

mg of sample was used for 2H NMR analysis, and this indicated ca. 2% deuterium

enrichment at both 84.4 and 85.0.

55

Cell-free Extract from S. capreolus

Preparation and Partial Purification of Cell-free Extract from S. capreolus

Buffer A: 10 mM KH2PO4, 10 mM K2HPO4, 5 mM dithiothreitol (DTT) and

15% glycerol (v/v), pH 7.5.

Buffer B: buffer A + 1mM EDTA.

All operations were carried out at 4 °C.

Step 1: Cells from 200 mL of a 48 h fermentation of S. capreolus were harvested

by centrifugation (4 °C, 13800 g, 20 min), and then washed (200 mL each) sequentially

with buffer A, 1.0 M KC1, 0.8 M NaC1, and buffer A. After each wash the cells were

centrifuged as above. The washed cells (22 g, wet wt.) were then resuspended in buffer B

(40 mL), and phenylmethanesulfonyl fluoride (PMSF) (16 mg in 1 mL isopropanol) was

subsequently added. The cells were then disrupted by sonication (power level 7 with

micro-tip, 80% duty cycle, pulsed for 6 x 20 s in total 12 min). Cell debris was removed

by centrifugation (4 °C, 13400 g, 20 min), and the supernatant was decanted to afford a

crude cell-free extract (CFE, 45 mL).

Step 2: Polyethylenimine (PEI) cellulose (Sigma, 50% in water, w/w) was added

to the CFE to a final concentration of 0.05%. After gentle stirring for 20 min, the cloudy

solution was centrifuged (4 °C, 13400 g, 20 min) and the precipitate was discarded.

Step 3: The supernatant was brought to 45% saturation by addition of solid

ammonium sulfate (Sigma, Grade III). The suspension was stirred for 30 min, and the

precipitate was collected by centrifugation (4 °C, 13400 g, 20 min) and stored at -80 °C.

The resulting supernatant was brought to 60% saturation with solid ammonium sulfate

and was stirred for additional 30 min. The pellet was obtained by centrifugation (4 °C,

13400 g, 20 min) and stored at -80 °C. The 60% (NH4)2SO4 saturated supernatant was

further brought to 80% saturation with solid ammonium sulfate and was stirred for 30

56

min. The pellet was collected by centrifugation (4 °C, 13400 g, 20 min) and stored at -80

°C.

Step 4: Buffer B (2 mL) was added to each of the three pellets which had been

stored at -80 °C for 12 h, and the dissolved pellets were loaded onto PD-10 spin-columns

packed with ca. 5 mL of Sephadex G-25 (pre-equilibrated in buffer B). The elutants were

transferred to three vials. The columns were washed once with 1 mL of buffer B. The

washings were combined with the corresponding elutants and used for enzyme activity

assays.

Protein Concentration Determination

Samples to be assayed were prepared as follows: to an 1.5-mL microfuge tube,

200 1.1.1, of the dye reagent and 800 RI, of protein solution (in most cases, a diluted

solution) were mixed and vortexed. After standed for 5 min, the samples were measured

at 595 nm by an UV-spectrophotometer individually, and protein concentrations were

determined by a standard calibration curve prepared from BSA (Figure 22).

Enzyme Activity Assays

The amino acid activating enzyme activity was assayed using an ATP -

[32P]pyrophosphate exchange reaction. The assay mixture contained 10 mM phosphate

buffer, pH 7.5, 2.5 mM ATP, 4.0 mM MgC12, 5.0 mM dithiothreitol (DTT), 1.0 mM

EDTA, 10 mM substrate, 50 1.11. [32P]pyrophosphate (5.15 x 105 dpm), and 250 pi.

enzyme solution, and incubated at 32 °C for 30 mM.

Trifluoroacetic acid (100 pl. of 10% ) was added to quench the exchange reaction

and the proteins were removed by centrifugation (7000 rpm, 2 min). The supernatant was

transferred to new tubes followed by addition of 50 µL of charcoal suspension (3% in 0.1

M Na4P207). The mixture was then vortexed, centrifuged, and the supernatant was

removed. The remaining charcoal residue was washed three times with water (3 x 800

57

!IL) and centrifuged each time. Finally, 100 pL of 10% aqueous pyridine was added to

the charcoal, and the mixture was vortexed and centrifuged at 7000 rpm for 4 min. A

portion of this solution (50 ilL) was used for LSC.

The rest of the solution was used for autoradiographic study. A PEI-cellulose TLC

plate was spotted with ATP first and then with the solution (50 gL), and developed in 0.5

M LiC1 solution. The air-dried plate was then exposed to a Kodak X-ray film in a light-

resistant cassette. The cassette was placed at -80 0C for 12 h before development of the

film.

58

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