Vol. 171, No. 5

Benzaldehyde Lyase, a Novel Thiamine PPi-Requiring Enzyme,from Pseudomonas fluorescens Biovar I

BERNARDO GONZALEZ AND RAFAEL VICUNA*

Laboratorio de Bioquimica, Unidad de Microbiologia y Genetica Molecular, Universidad Cat6lica de Chile,Casilla 114-D, Santiago, Chile

Received 15 September 1988/Accepted 26 January 1989

Pseudomonasfluorescens biovar I can grow on benzoin as the sole carbon and energy source. This ability isdue to benzaldehyde lyase, a new type of enzyme that irreversibly cleaves the acyloin linkage of benzoin,producing two molecules of benzaldehyde. Benzaldehyde lyase was purified 70-fold and found to requirecatalytic amounts of thiamine PP; (TPP) and a divalent cation as cofactors. Optimal activity was obtained witha 1.0 mM concentration of Mn2 , Mg2+, or Ca2'. Gel permeation chromatography indicated a nativemolecular weight of 80,000, whereas the enzyme migrated in sodium dodecyl sulfate-containing polyacrylamidegels as a single polypeptide with a molecular weight of 53,000. Benzaldehyde lyase is highly specific; of a varietyof structurally related compounds tested, only benzoin and anisoin (4,4'-dimethoxybenzoin) acted as

substrates, their apparent K14s being 9.0 x 10-3 and 3.25 x 10-2 mM, respectively. A catalytic mechanism forthe enzyme is proposed.

Pseudomonasfluorescens biovar I, a strain we isolated inenrichment cultures containing the lignin model compoundanisoin as the sole carbon and energy source, quantitativelydegrades this substrate, producing anisic acid as a metabolicintermediate (5, 6). When crude extracts from this bacteriumwere prepared, they catalyzed the conversion of benzoin andanisoin to benzaldehyde and anisaldehyde, respectively, inthe absence of any added cofactor (6). However, we foundlater that this activity was lost upon extensive dialysis or

fractionation of this extract and that it could be restored onlyby addition of thiamine PP1 (TPP) and a divalent cation salt.

Several TPP-requiring enzymes have been described. Oneof the most widely known is transketolase, which transfers a

glycolaldehyde moiety from a phosphorylated ketose to a

phosphorylated aldose acceptor (9).Other examples of TPP-requiring enzymes are the follow-

ing, which are involved in the metabolism of pyruvate.Pyruvate oxidase catalyzes the oxidative decarboxylation ofthis three-carbon acid to form acetate (21). Pyruvate dehy-drogenase, an enzymatic complex, decarboxylates pyruvateto produce acetyl coenzyme A (16). Pyruvate decarboxylasecatalyzes the decarboxylation of pyruvic acid to yield acet-aldehyde (12). Acetohydroxy acid synthase forms acetolac-tate, a precursor of valine, from two molecules of pyruvate(7). All these reactions proceed through a common interme-diate: 2-hydroxyethyl-TPP (12). Due to its high reactivity,enzymes from higher plants and yeast have been found totransfer this two-carbon unit to an aldehyde receptor to forma ketol. This is the so-called acyloin condensation or carbo-ligase reaction (10).

Acetoin dehydrogenase and benzoylformic decarboxylasealso require TPP. The former, described for Bacillus subtilis,catalyzes the cleavage of the ketol acetoin to give acetalde-hyde plus acetate (13). The latter, found in Pseudomonasputida, produces benzaldehyde plus CO2 from benzoylfor-mic acid (8).

In the present study, we describe the purification andcharacterization of benzaldehyde lyase from P. fluorescens

* Corresponding author.

biovar I. This enzyme cleaves the a-hydroxy ketones ben-zoin and anisoin in reactions that require TPP and a divalentcation (Fig. 1). To our knowledge, this represents a noveltype of TPP-requiring enzyme.

MATERIALS AND METHODS

Strain. The isolation of P. fluorescens biovar I from a pulpmill effluent has been described previously (5).

Chemicals. The following chemicals were used (romannumerals refer to Fig. 2): anisic (4-methoxybenzoic) acid,anisaldehyde (4-methoxybenzaldehyde), benzoin (2-hydroxy-1,2-diphenylethanone [compound I], anisoin [2-hydroxy-1,2-bis(4'-methoxyphenyl)-ethanone [compound I'], desoxyben-zoin (1,2-diphenylethanone [compound II]), desoxyanisoin[1,2-bis(4'-methoxyphenyl)-ethanone (compound II')], ben-zoin ethyl ether (2-ethoxy-1,2-diphenylethanone [compoundV]), 2,2-dimethoxy-1,2-diphenylethanone (compound VI),dimethoxybenzil [1,2 - bis(4' - methoxyphenyl) - ethanedione(compound VIII)], benzoin oxime (2-hydroxy-1-isonitroso-1,2-diphenylethanone [compound IX]), a-pyridoin [2-hydroxy-1,2-bis(2-pyridyl)-ethanone (compound X)], and furoin [2-hydroxy-1,2-bis(2-furyl)-ethanone, (compound XI)]. All were

purchased from Aldrich Chemical Co., Inc. Dihydrobenzoin(1,2-diphenyl-1,2-ethanediol [compound VII]) and dihy-droanisoin [1,2-bis(4'-methoxyphenyl)-1,2-ethanediol (com-pound VII')] were obtained by quantitative reduction ofbenzoin and anisoin, respectively (6). The synthesis of 3-hydroxy-1,2-diphenyl-propanone (compound III) and 3-hy-droxy-1,2-bis(4'-methoxyphenyl)-propanone (compound III')from desoxybenzoin and desoxyanisoin, respectively, was

performed by the method of Kirk and Nakatsubo (11).Anisoin was acetylated with acetic anhydride to obtain 2-acetoxy-1,2-bis(4'-methoxyphenyl)-ethanone (compound IV).1-[3,4-Dimethoxyphenyl]-2,3-dihydroxypropanone (compoundXII) was a generous gift of K. Kirk from the Forest ProductsLaboratory, Madison, Wis. All these aromatic compoundswere kept in stock solutions (400 mg/ml) in N,N'-dimethyl-formamide. TPP, acetoin [3-hydroxy-2-butanone] (com-pound XIII), and benzoylformic (2-oxo-phenylethanoic) acid

2401

JOURNAL OF BACTERIOLOGY, May 1989, p. 2401-24050021-9193/89/052401-05$02.00/0Copyright ©D 1989, American Society for Microbiology

on May 13, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2402 GONZALEZ AND VICUNA

TPP

Me2

O H

%C20

R =H or OCH3

FIG. 1. Reaction catalyzed by benzaldehyde lyase. Me2+ refersonly to the metal ions Mg2+, Mn2+, and Ca2+.

were obtained from Sigma Chemical Co., Milwaukee, Wis.All the other reagents and solvents were analytical grade.

Purification of benzaldehyde lyase from P. fluorescens bio-var I. For the preparation of enzyme, the bacterium was

grown at 30°C with agitation in 2.0-liter Erlenmeyer flasksfilled with 1.0 liter of minimal medium containing 1.0 mg ofanisoin per ml and harvested in early stationary phase. Allthe following operations were carried out at 4°C. Cells (30 g)were suspended in 90 ml of a buffer containing 20 mM Trishydrochloride (pH 8.0), 1.0 mM EDTA, 5.0 mM 2-mercap-toethanol, and 20% glycerol and disrupted by sonication.The extract was centrifuged in a rotor (JA-20; BeckmanInstruments, Inc., Fullerton, Calif.) at 17,640 x g for 20 min.In order to separate nucleic acids, the supernatant was made5.0% in streptomycin sulfate, kept on ice for 20 min, andspun for 10 min at 7,840 x g in a JA-20 rotor. Proteins were

concentrated from the supernatant by precipitation with 60%ammonium sulfate. After centrifugation at 31,360 x g for 30min in a Beckman JA-20 rotor, the pellet was suspended inthe buffer described above to a final protein concentration of10 to 15 mg/ml and dialyzed against 1 liter of a buffercontaining 20 mM Tris hydrochloride (pH 8.0), 5.0 mM2-mercaptoethanol, 0.05 mM TPP, 1.0 mM MgCl2, and 20%glycerol (buffer A) for 4 to 6 h. This crude extract was thenloaded onto a DEAE-cellulose column (45 cm by 4.2 cm2)equilibrated in buffer A. The column was thoroughly washedwith 200 mM KCl dissolved in buffer A and the activity waseluted with a 600-ml linear salt gradient (200 to 500 mMKCl). Five-milliliter fractions were collected. Benzaldehydelyase eluted at 250 mM KCl. The active fractions were

pooled (60 ml) and dialyzed for 2 to 3 h against buffer B,which consisted of 20 mM phosphate (pH 6.5), 5.0 mM

RI

H(OCH3)

H IOCH3)I (I) Ri=OH.R2=H

11 (11) R, =R2-HIll (Ill'): R, =H R2=CH20Hl' .R,I= IICC ,R2=HOCCH3

V R1 = OCH2CH3 .R2-H

VI R-R2= OCH3

OH

0°e~R249 R,

N

X R,=2-"R2-<

XI.IR=R2=

Xll: R,= < .R2CH120H)C3

OCH3

XIII1 Ri=R2= CH3

IxFIG. 2. Structures of compounds tested as possible substrates of

benzaldehyde lyase. Their names are listed in Materials and Meth-ods.

2-mercaptoethanol, 0.05 mM TPP, 1.0 mM MgCl2 and 20%glycerol. This pool was then loaded onto a hydroxylapatitecolumn (HTP; Bio-Rad Laboratories, Richmond, Calif.) (13cm by 15 cm2) equilibrated in buffer B. The protein waseluted with 1.2 liters of a linear phosphate (pH 6.5) gradient(20 to 200 mM), and 5.2-ml fractions were collected. Theenzyme eluted at 50 mM phosphate. Active fractions werepooled (110 ml), and the enzyme was concentrated byadsorption to a 0.5-ml DEAE-cellulose column. It was elutedwith 0.5 ml of buffer A containing 1.0 M KCl and subjectedto gel permeation chromatography in an Ultrogel AcA-34column (85 cm by 2.5 cm2) equilibrated with buffer A plus 0.3M KCl; 1.5-ml fractions were collected. Active fractionswere pooled (15 ml), diluted twofold in buffer A, andconcentrated in a DEAE-cellulose column as describedabove. This final preparation was dialyzed against buffer Aand stored at -20°C. Under these conditions, benzaldehydelyase was stable for at least 2 months.Enzyme assays. (i) Benzaldehyde lyase. One-milliliter solu-

tions containing 0.15 mM substrate, 20 mM Tris hydrochlo-ride (pH 8.0), 0.01 mM TPP, and 0.1 mM MgCl2 wereincubated at 37°C for 3 to 5 min. Reactions were started bythe addition of 1.0 to 5.0 enzyme units and further incubatedfor 2 min. Enzymatic activity was monitored spectrophoto-metrically at 250 nm with benzoin or at 280 nm with anisoin.Each substrate and its corresponding aldehyde product havesimilar absorbance maxima. However, since two moleculesof aldehyde are produced per each molecule of substratecleaved, the following formula was used to relate absorbancechange to product formed (see Appendix): nanomoles ofproduct formed =[2(Ai - Af)I(E, - 2£p)] x 106, with es andrp being the molar extinction coefficients of the substrate andthe product. Ai - Af is the change in optical density duringthe reaction time, measured at either 250 or 280 nm. Molarextinction coefficients determined experimentally in aqueoussolutions at neutral pH for benzoin (250 nm), benzaldehyde(250 nm), anisoin (280 nm), and anisaldehyde (280 nm) were12,670, 11,500, 11,000, and 9,500, respectively. One unit ofenzymatic activity was defined as the amount of enzymenecessary to produce 1 nmol of aldehyde per min under theabove conditions.

(ii) Activity with substrate analogs. Enzyme assays wererun in cuvettes containing 0.05 to 0.2 mM concentrations ofthe aromatic compound prepared in the incubation medium,and spectral changes were measured at an appropriatewavelength (250, 280, or 310 nm). In each case, reactionswere analyzed by thin-layer chromatography after incuba-tion for 30 min. For testing acetoin (Fig. 2, compound XIII)as the substrate, the medium was supplemented with alcoholdehydrogenase plus NADH (Sigma) and the reaction wasmonitored at 340 nm.

(iii) Inhibition assays. The effect of substrate analogs onthe activity of benzaldehyde lyase was studied under theconditions described above, except that reaction mixturescontained 0.15 to 0.2 mM of a substrate analog and werepreincubated with the enzyme for 5 min before benzoin oranisoin was added.Other procedures. Polyacrylamide gel electrophoresis un-

der native conditions was carried out by the method of Davis(4), with both the gel and running buffers containing 0.04 mMTPP and 1.0 mM MgCl2. For determination of enzymaticactivity after electrophoresis, gels were cut into 2.0-mmslices. Each slice was submerged in 0.3 ml of buffer A, andafter overnight incubation at 4°C, samples were withdrawnto be assayed for activity. Electrophoresis in gels containingsodium dodecyl sulfate was performed as described by

J. BACTERIOL.

on May 13, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

NOVEL TPP-REQUIRING ENZYME FROM P. FLUORESCENS

TABLE 1. Summary of the purification procedure

Ttl TotalPui-nTotal enzyme Sp act Yield Purjfi-protein catioFraction activity (U/mg) (% (fold(mg) (U) (od

Crude extract 86.8 14,714 170 100.0 1.0DEAE-cellulose chroma- 3.6 11,250 3,125 76.5 18.4

tographyHydroxylapatite chroma- 1.42 7,219 5,084 49.0 29.9

tographyUltrogel AcA-34 chroma- 0.43 5,025 11,687 34.1 68.7tography

Weber and Osborn (19) with slab gels (7 by 10 by 0.05 cm).Protein was stained with Coomassie brilliant blue. High-performance liquid chromatography and thin-layer chroma-tography' were carried out as previously reported (6). Proteinconcentrations were determined by the method of Bradford(1).

RESULTS AND DISCUSSION

Purification of the enzyme. By following the isolationprocedure summarized in Table 1, a 70-fold purification ofbenzaldehyde lyase from P. fluorescens biovar I was ob-tained. Samples obtained after each purification step were

subjected to polyacrylamide gel electrophoresis in the pres-ence of sodium dodecyl sulfate (Fig. 3A to C). Enzymeobtained after Ultrogel AcA-34 chromatography was usedfor all the experiments described in this report. When thispreparation was subjected to polyacrylamide gel electropho-resis run under nondenaturing conditions, activity could berecovered from the gel. However, the yields were too low toallow its utilization in these studies. Affinity chromatographywas attempted at various stages of enzyme purification witheither anisic acid or TPP as the ligand. They were linked to

A B C D

kd..kd W ."...W.

as

45

35 -

29

24

20.1

14.2-

FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of protein samples after (A) DEAE-cellulose, (B) hydroxylap-atite, and (C) Ultrogel AcA-34 chromatography and (D) nativepolyacrylamide gel electrophoresis. Numbers on the left refer to themolecular masses (in kilodaltons [kdl) of protein standards (DaltonMark VII; Sigma).

AH-Sepharose 4B by the carbodiimide coupling procedure(3, 15). Benzaldehyde lyase did not bind to TPP-linkedSepharose. In contrast, it did bind to anisyl-Sepharose,although after the column was washed with 1.0 M KCI andthen eluted with 0.3 M anisic acid, no significant incrementin specific activity was observed.

Molecular weight estimation. Ultrogel Aca-34 filtrationshowed an apparent molecular weight of 80,000. For thisdetermination, human immunoglobulin G (150,000), bovineserum albumin (66,000), ovalbumin (45,000), chymotrypsin-ogen A (24,300) and lysozyme (12,300) were utilized 'asmolecular weight standards to calibrate the column. In turn,benzaldehyde lyase eluted in the excluded volume of aSephadex G-75 column. The activity recovereq from thenative gel mentioned above was also subjected to electro-phoresis in the presence of sodium dodecyl sulfate. Underthese conditions it showed a single band with a molecularweight of 53,000 (Fig. 3D), suggesting that the native enzymemay be either a dimer or a single polypeptide with anonspherical shape.Requirements for activity. (i) TPP. The enzyme was totally

inactive in the absence of TPP. Enzyme activity could bedetected spectrophotometrically at a concentration of TPPas low as 10 nM, showing that this cofactor acts in a catalyticfashion. Maximal activity was found at 0.01 mM TPP, whileconcentrations higher than 0.5 mM were inhibitory. Depen-dence on TPP for activity is less pronounced in crudeextracts (6), indicating that the cofactor remains attached tothe enzyme after ammonium sulfate precipitation. However,extensive dialysis or DEAE-cellulose chromatography of theextract sufficed to inactivate benzaldehyde lyase unless TPPwas added to the assay mnixture. This is in contrast to otherTPP-requiring enzymes, which bind the cofactor in a non-dissociating manner (14). When thiamine hydrochloride wasadded instead of TPP, no reactivation occurred.

(ii) Metal ions. In early experiments, divalent cations werenot added to the enzymatic assays (6). However, we soonnoticed that the same enzyme preparation behaved differ-ently in the presence of a metal ion. This observationprompted us to detertnine the effect of divalent cations in thereaction. For this purpose, assay mixtures were made with0.06 mM EDTA. Under these conditions, benzaldehydelyase is completely inactive. Full activity was obtained witha 1.0 mM concentration of MgCl2, MnSO4, or CaSO4. At 5.0mM, activities were about 50% of the optimal for each ofthese cations. Activity was not restored with 1.0 mMCoS04, ZnSO4, NiSO4, FeSO4, or A12(SO4)3, and it waspartially recovered with 1.0 mM CuSO4 or BaSO4. Thefollowing monovalent ions at 1.0 to 160 mM were neitheractivators nor inhibitors: KCI, NaCl, and NH4Cl. All thesedeterminations were made in the presence of 0.01 mM TPP.Requirement for a divalent cation constitutes a commonfeature of most enzymes utilizing TPP, e.g., pyruvate decar-boxylase (14), at-ketoarginine decarboxylase (18), spinachtransketolase (9), and acetohydroxy acid synthase (7), al-though optimal cpncentrations have not been reported. Onthe other hand, no requirement for a metal cofactor(s) hasbeen described for liver transketolase (9) or benzoylformicdecarboxylase (8).

Substrate specificity. A series of aromatic ketols weretested as possible substrates of the enzyme. In addition, todetermine the effect of chemical modifications in the acyloinlinkage, we also tested desoxy- (Fig. 2, compou'nds II andII'), hydroxymethyl- (compounds III and III'), acetylated(compound IV), ethylated (compound V) and methylated(compound VI) derivatives of anisoin or benzoin. The diolic

VOL. 171, 1989 2403

on May 13, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2404 GONZALEZ AND VICUNA

(compounds VII and VII'), dibenzal (compound VIII), andoxime (compound IX) forms of benzoin or anisoin were alsoassayed. Of all these compounds, only benzoin (compoundI) and anisoin (compound I') were cleaved by benzaldehydelyase. Both reactions proceeded to completion, since nosubstrate could be found after prolonged incubation. Appar-ent K,,s for compounds I and I' were 9 x 10-' and 3.25 x10-2 mM, respectively. In turn, no conversion of benzalde-hyde or anisaldehyde to benzoin or anisoin could be detectedby either spectrophotometry, thin-layer chromatography, orhigh-performance liquid chromatography. Furthermore, atconcentrations up to 0.2 mM, the aldehydes failed to inhibitthe enzyme, indicating that benzaldehyde lyase catalyzes anirreversible reaction. This implies that the enzyme is unableto form the benzoyl-TPP intermediate (see below) startingfrom the aldehyde. Another possibility is that the interme-diate is indeed formed but that it decomposes 8lue to its highinstability, as is the case with the chemically synthesizedcompound (2). Our attempts to synthesize benzoyl-TPP atalkaline pH starting from TPP and benzaldehyde were alsounsuccessful.Compounds II, II', IV, and V (Fig. 2) at concentrations of

0.9, 0.4, 0.2, and 0.9 mM, respectively, produced an averageinhibition of 50%, suggesting that they are somehow recog-nized by the enzyme. The nature of this inhibition was notfurther studied due either to the low solubility of thesearomatic compounds in water or to optical interference.Other compounds, such as benzoylformic acid (0.9 mM),pyruvic acid (10 mM), and acetoin (10 mM), did not inhibitthe enzyme.These results indicate that benzaldehyde lyase is highly

specific. P. fluorescens biovar I cells do not express benz-aldehyde lyase activity when they are grown on glucose orrich media either in the presence or in the absence of anisoin.However, enzyme with lower specific activity (20 to 40%)was detected in the extract of cells grown on benzoylformicor benzoic acid. We were not able to detect this enzymaticactivity in crude extracts from P. putida KT 2440 grown onbenzoic acid or P. putida KT 2440 and Escherichia coli HB101 grown on rich media. These two strains do not metabo-lize anisoin. Since benzoin or anisoin are not expected to bewidespread in nature, the physiological role of this enzymein P. fluorescens biovar I remains to be established.

Catalytic mechanism. Considering the available informa-tion about reactions involving TPP (12, 17, 20) plus the dataobtained in the present study, we propose for the reactioncatalyzed by benzaldehyde lyase the mechanism schema-tized in Fig. 4. First, the ilide form of TPP attacks thecarbonyl carbon atom of the ot-hydroxy ketone substrate toproduce an adduct. The highly electrophilic nitrogen atom ofthe TPP moiety promotes an electron rearrangement leadingto the formation of the enamine intermediate benzoyl-TPPand the first molecule of free aldehyde. Protonation of thisintermediate then releases the second molecule of aldehydeand restores the cofactor. As indicated above, these stepsare probably irreversible since benzaldehyde or anisalde-hyde neither forms benzoyl-TPP directly nor inhibits theformation of benzoyl-TPP from the ketol.The first half of the overall reaction, up to the formation of

one of the aldehyde molecules, is similar to those of tran-sketolase and acetoin dehydrogenase. However, the mech-anism for the resolution of the enamine intermediate byaddition of an electrophilic reagent differs in these threeenzymes. In the case of transketolase, the intermediate un-dergoes the addition of a phosphorylated aldehyde to generatea phosphorylated ketose (9, 12). With acetoin dehydrogenase,

I HSHO-cH-2

R'-CHO

c o> HO-C

I ~~~~~~R

N,,,CH2 /CH2

FIG. 4. Proposed catalytic mechanism for benzaldehyde lyase.First, the carbonyl carbon of the substrate is attacked by the ilideform of TPP, forming an adduct with a tetrahedral carbon. Thehighly electrophilic nitrogen directs an electron rearrangement,leading to the formation of an enamine intermediate and the releaseof an aldehyde molecule. The former is attacked by a proton, which

produces another electron flow that releases the second aldehydemolecule plus the ilide.

the intermediate is oxidized and produces acetate plus TPP(13). In the case of benzaldehyde lyase, the intermediate isprotonated to give aldehyde plus TPP. This second part of thereaction is equivalent to that of TPP-requiring decarboxylases(12, 20). In all of them, release of aldehyde takes place afterattack of the enamine adduct by a proton.

Other properties of benzaldehyde lyase. The enzyme showsmaximal activity between pH 7.5 and 8.5, whereas it isinactive below pH 6.0. For most determinations, two or threedifferent buffers were used and similar results were obtained.No sulfhydryl groups are required for activity since the

enzyme is neither inhibited by 20 mM N-ethylmaleimide nor

activated by 5.0 mM dithiothreitol. Benzaldehyde lyase isinactivated by 4.0 M urea but is refractory to treatment with0.1% sodium dodecyl sulfate or 10% ethanol.

APPENDIX

The following stoichiometry of the reaction for the formation of

aldehyde holds true:

concentration of aldehyde formed (N) =

2(initial concentration of substrate [Ci] -

final concentration of substrate [C,])According to Beer's law. the initial absorbance (Ai) can be

expressed as

(1)

J. BACTERIOL.

on May 13, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

NOVEL TPP-REQUIRING ENZYME FROM P. FLUORESCENS

Ai= Cj x es (2)

Since the absorbances are additive, the final absorbance is

Af=(C P(3)AS=(C X S) + N x Fp 3

where rs and sr, are the molar extinction coefficients of thesubstrate and the product, respectively.

If Ci is expressed as a function of Ai (from equation 2) and Cf isexpressed as a function ofAfand N (from equation 3 by replacementin equation 1) we obtain N = 2[(AI/s) - (A/,e)+N(rp,Ir)]. Byrearrangement, this equation becomes N = 2(Ai - Af)/(Es - 2Ep).Since the volume of 1.0 ml is constant, molar concentration can beconverted to nanomoles of product directly as follows: nanomoles ofproduct = [2(Ai - Af)/(Es - 2rp)] X 106.

ACKNOWLEDGMENTS

We are grateful to Jaime Eyzaguirre for critical reading of themanuscript.

This work was supported by Celulosa Arauco y Constituci6n,FONDECYT, and National Science Foundation grant no. 144X753.

LITERATURE CITED

1. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

2. Breslow, R. J. 1958. Mechanism of thiamine action. IV. Evi-dence from studies on model systems. J. Am. Chem. Soc.80:3719-3726.

3. Cuatrecasas, P., and C. B. Anfinsen. 1971. Affinity chromatog-raphy. Methods Enzymol. 22:345-378.

4. Davis, B. J. 1964. Disc electrophoresis. II. Method and applica-tion to human serum proteins. Ann. N. Y. Acad. Sci. 121:404-427.

5. GonzAlez, B., A. Merino, M. Almeida, and R. Vicuna. 1986.Comparative growth of natural bacterial isolates on various

lignin-related compounds. Appl. Environ. Microbiol. 52:1428-1432.

6. Gonzillez, B., I. Olave, I. Calder6n, and R. Vicuna. 1988.Degradation of diarylethane structures by Pseudomonas fluo-rescens biovar I. Arch. Microbiol. 149:389-394.

7. Grimminger, H., and H. E. Umbarger. 1979. Acetohydroxy acid

synthase I of Escherichia coli: purification and properties. J.Bacteriol. 137:846-853.

8. Hegeman, G. D. 1970. Benzoylformate decarboxylase (Pseudo-monas putida). Methods Enzymol. 17a:674-678.

9. Horecker, B. L., P. Z. Smyrniotis, and H. Klenow. 1953. Theformation of sedoheptulose phosphate from pentose phosphate.J. Biol. Chem. 205:661-682.

10. Juni, E., and G. A. Heym. 1956. Acyloin condensation reactionsof pyruvic oxidase. J. Biol. Chem. 218:365-378.

11. Kirk, T. K., and F. Nakatsubo. 1983. Chemical mechanism of animportant cleavage reaction in the fungal degradation of lignin.Biochim. Biophys. Acta 756:376-384.

12. Krampitz, L. 0. 1969. Catalytic functions of thiamin diphos-phate. Annu. Rev. Biochem. 38:213-240.

13. L6pez, J. M., B. Thoms, and H. Rehbein. 1975. Acetoin degra-dation in Bacillus subtilis by direct oxidative cleavage. Eur. J.Biochem. 57:425-430.

14. Morey, A. V., and E. Juni. 1970. Resolution, reconstitution, andother methods for the study of binding of thiamine pyrophos-phate to enzymes. Methods Enzymol. 18a:238-245.

15. O'Brien, T. A., H. L. Schrock, P. Russell, R. Blake II, and R.Gennis. 1976. Preparation of Escherichia coli pyruvate oxidaseutilizing a thiamine pyrophosphate affinity column. Biochim.Biophys. Acta 452:13-29.

16. Reed, L. J. 1974. Multienzyme complexes. Acc. Chem. Res.7:40-46.

17. Ulirich, J., Y. M. Ostrovsky, J. Eyzaguirre, and H. Holzer. 1971.Thiamine pyrophosphate-catalyzed enzymatic decarboxylationof 2-oxoacids. Vitam. Horm. 28:365-398.

18. Vanderbilt, A. S., N. S. Gaby, and V. W. Rodwell. 1975.Intermediates and enzymes between cx-ketoarginine and -y-guanidinobutyrate in the L-arginine catabolic pathway of Pseu-domonas putida. J. Biol. Chem. 250:5322-5329.

19. Weber, K., and M. Osborn. 1971. The reliability of molecularweight determinations by dodecyl sulfate polyacrylamide. J.Biol. Chem. 244:4406-4412.

20. Weiss, P. M., G. A. Garcia, G. L. Kenyon, W. W. Cleland, andP. F. Cook. 1988. Kinetics and mechanism of benzoylformatedecarboxylase using 13C and solvent deuterium isotope effectson benzoylformate and benzoylformate analogues. Biochemis-try 27:2197-2205.

21. Williams, F. R., and L. P. Hager. 1966. Crystalline flavinpyruvate oxidase from Escherichia coli. Arch. Biochem.Biophys. 116:168-176.

2405VOL. 171, 1989

on May 13, 2020 by guest

http://jb.asm.org/

Dow

nloaded from