THE JOURNAL OF BIOL~~ICAL No. 40, 7, pp. 1994 by The … · 2001-06-29 · Structure and Mechanism...

T H E JOURNAL OF BIOL~~ICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 40, Issue of October 7, pp. 25095-25105, 1994 Printed in U.S.A.

Structure and Mechanism of Galactose Oxidase THE FREE RADICAL SITE*

(Received for publication, May 23, 1994)

Andrew J. Baron, Conrad Stevens*, Carrie Wilmot, Kaqjula D. Seneviratnes, Veronica Blakeley, David M. Dooleyfl, Simon E. V. Phillipsll, Peter F. Knowles, and Michael J. McPherson** From the Department of Biochemistry and Molecular Biology, The University of Leeds, Leeds, LS2 SJT, United Kingdom and the Wepartment of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 5971 7-0340

Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordi- nated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild- type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. Afurther variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.

Galactose oxidase (EC 1.1.3.9) is an extracellular enzyme produced by a Fusarium spp.’ and is the simplest known copper-containing oxidase. Its structure and catalytic mechanism have been studied extensively using biochemical and spectroscopic methods (for review see Knowles and Ito, 1993). The x-ray structure to 1.7 h resolution has been reported for a crystal form at pH 4.5 (Ito et al., 1991). In addition the gaoA gene that encodes galactose oxidase has been characterized (McPherson et al., 1992b).

Galactose oxidase catalyzes the stereospecific oxidation of D-isomers of a range of primary alcohol substrates, including D-galactose and polysaccharides with D-galactose at their non- reducing end, leading to the production of the corresponding

* This work was funded in part by the United Kingdom Science and Engineering Research Council. The costs of publication of this article

therefore be hereby marked “aduertisenent” in accordance with 18 were defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Medical Research Council Research Studentship.

Present address: Dept. of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 2RB, U. K.

8 Recipient of an SERC Research Studentship. Present address: School of Biological Sciences, Stopford Building, University of Manches- ter, Manchester M13 9PL, U. K.

and International Research Scholar of the Howard Hughes Medical 11 Science and Engineering Research Council Senior Research Fellow

Institute. ** Leverhulme Trust/Royal Society Senior Research Fellow. To whom

correspondence should be addressed. %I.: 44-532-332-595; Fax: 44-532- 333-144; E-mail: [email protected].

referred to throughout the literature as D. dendroides (or Cladobotryum The organism that produces the well studied galactose oxidase is

dendroides). Recently this organism, strain 2903 from the Northern Regional Research Laboratory (NRRL; Peoria, IL), has been shown to be a Fusarium spp. (Ogel et al., 1994), a finding of importance in the context of defining the biological function of this extracellular oxidase.

aldehyde and hydrogen peroxide. The enzyme is a monomer comprising three domains with a single Cu(I1) atom close to the surface in domain 2. In the crystal form at pH 4.5 the copper site (Fig. 1) is essentially square pyramidal with w7’, His496, His518, and an acetate ion, as equatorial ligands and T y r 4 ” as the axial ligand. Most interestingly, a novel covalent thioether bond between the Sr of CysZz8 and CE of Q + I 2 has been identified in the crystal structure and confirmed by other studies (Ito et al., 1991; Knowles and Ito, 1993). The extended aromatic region resulting from the coplanarity of the side chains of w7’ and Cys228 is involved in an aromatic stacking interaction with TrpZg0 (Ito et al., 1991,1994). Extensive spectroscopic data have been presented to support the proposal that galactose oxidase uses a free radical mechanism for catalysis (Whittaker and Whittaker, 1988, 1993; Whittaker et al., 1989). When consid- ered in the light of the crystal structure, the spectroscopic results suggest that w7’ is a component of the radical site. The spectroscopic characteristics of galactose oxidase in the visible region suggest that the intense peak at 445 nm arises from overlapping ligand-to-metal charge transfer between tyrosine and copper and rr + rr* ring transitions in the tyrosine radical. The “red” peak at 810 nm has been assigned to a mixture of ligand-to-metal charge transfer and charge resonance excitation between aromatic n-systems involved in the charge transfer complex (Whittaker et al., 1989; Whittaker and Whittaker, 1993).

Site-directed mutagenesis, in conjunction with structural and mechanistic characterization of the variant proteins, represents a powerful approach to understanding enzyme catalytic processes including those involving protein free radicals (Ormo et al., 1992). To assess how active site amino acid residues contribute to the catalytic process and to the formation and stabilization of the radical species, we have generated mutational variants of w7‘, CysZz8, and TrpZg0. We present crystallographic, visible spectroscopic, and enzyme kinetic data for native, recombinant heterologously expressed wild-type, and the variant forms of galactose oxidase. The data are discussed in terms of our current understanding of the active site derived from crystallographic and spectroscopic studies.

EXPERIMENTAL PROCEDURES Strains and Vectors

Escherichia coli strains DHl (F, gyrA96, recA1, endA1, thi-1, hs- (IRK-, hsd MK’, supE44; Hanahan, 1983) and its derivative XL1-Blue (Stratagene), which carries the additional markers (relAl, lac-,[F, prom, lacIq.ZDM15, TnlO(tetTl were used.

Aspergillus nidulans strain G191 (pabaA1, pyrG89, fwal, uaY9), kindly supplied by Prof. G. Turner (University of Sheffleld, U. K.), is unable to grow in the absence of exogenous uracil because of a mutation in the pyrG gene, which encodes the enzyme orotidine-5‘-phosphate carboxylase.

25095

25096 Structure and Mechanism of Galactose Oxidase

L Tyr495

72

cg5

Trp290 Trp290

oxidase crystal structure at pH 4.5. CysZ2* and the stacked TrpzS0 are FIG. 1. Stereo view of the copper coordination in the galactose

also shown.

pGAO9 is a pBluescript derivative with a 7.35-kb2 insert carrying the galactose oxidase gene (McPherson et al., 1992b) isolated from a filamentous fungus previously referred to as Dactyliurn dendroides, but recently reidentified as a Fusarium spp. (Ogel et al., 1994).

pGAO11 is a derivative of pGAO9 with an insert of 3,466 bp which includes the galactose oxidase coding region and for which the complete nucleotide sequence has been determined (McPherson et al., 1992b). pGAOll was constructed in this laboratory by Z. B. Ogel (1993).

pRG4, from Prof. G. May (Houston, TX), is a shuttle vector based on pBluescript which carries an ampicillin resistance gene for selection in E. coli and the Neurospora crussa pyr4 gene, the equivalent of pyrG of A. nidulans, allowing selection of A. nidulans G191 transformants by uracil prototrophy (Oakley et al., 1987).

pGPT-pyrG1, from Dr. M. Ward (Genencor, San Francisco, CA), is a shuttle vector carrying an ampicillin resistance gene for selection in E. coli and the pyrG gene of A. nidulans allowing uracil prototrophy selection in strain A. nidulans G191. In addition it carries the promoter region of the glucoamylase gene (glaA) ofAspegi1lu.s awamori, and the glaA terminator region of Aspergillus niger flanking unique BglII and XbaI cloning sites (Ward et al., 1990).

DNA Preparation and Manipulations Plasmid DNA was purified from E. coli cultures by the alkaline lysis

method (Sambrook et al., 1989) with additional purification for DNA sequencing by polyethylene glycol precipitation as described by Kraft et al. (1988). DNA sequencing of double-stranded plasmid DNA was performed using Sequenase version 2.0 (U. S. Biochemical Corp.), according to the manufacturer's instructions. Unless otherwise indicated, restriction enzymes and ligation reactions were performed according to the recommendations of the enzyme manufacturer and calf intestinal phosphatase treatment according to Sambrook et al. (1989). Southern blot hybridizations were performed as described previously (McPherson et al., 1992b). Oligonucleotides were synthesized on an Applied Biosys- tems 381Ainstrument and were used without further purification. DNA fragments were recovered from agarose gels by centrifuging the gel slice in a Spin-X filter unit (Costar; McPherson et al., 1992a) or were used directly in NuSieve agarose gel (FMC Bioproducts, Rockland, ME) for subsequent manipulations.

PCR-based Mutagenesis A procedure involving two PCRs was used to generate a DNA frag-

ment carrying the desired mutation. This "mutant" fragment was used to replace a corresponding region of the wild-type gene as illustrated in Fig. 3. The oligonucleotide sequences used for mutagenesis were

Y272F: 5'-CTCGTGGGTKCAGTCATC-3' C228G: 5'-GATATGTTCQCCTGGTATC-3' W290H: 5'-TGGAGGCTCC=AGCGGTGGCGT-3'

SEQUENCES 1-3

where the base changes in the mutagenic primers are underlined. The flanking primers a and b were selected from a bank of previously available DNA sequencing primers (McPherson et al., 1992b).

polymerase chain reaction; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline- The abbreviations used are: kb, kilobase(s); bp, base paids); PCR,

6-sulfonic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.

PCRl-A 50-pl reaction contained wild-type plasmid DNA template (50 ng), 5 pl of 10 x VentTM polymerase buffer containing 60 mM MgCI,, 1.5 p1 of 10 mM each dNTP, 50 pmol each of the mutagenic primer and primer a (see Fig. 3) and was overlaid with 50 pl of mineral oil. The temperature regime was 95 "C for 4 min and then 25 cycles of 95 "C for 1 min; 55 OC for 1 min; 72 "C for 1 mid. One unit of Vent polymerase (New England Biolabs) was added during the initial denaturation period.

The reaction mixture was added to 300 pl of 300 mM sodium acetate, 1 mM EDTA, pH 7.0, and the DNA precipitated by addition of 750 pl of ethanol a t -20 "C. The precipitate was collected by centrifuging, and the pellet was washed in 70% ethanol, air dried, and then resuspended in 8 p1 of TE (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) before loading on a 2% NuSieve agarose gel containing 1 pg/ml ethidium bromide. The desired band was excized under UV illumination and was soaked in 5 ml of double-distilled water for 30 min.

PCR2-The NuSieve gel from PCRl provided the source of megaprimer for PCR2; the agarose melted during the first 95 "C step and remained molten during PCR cycling. A 50-pl reaction contained 50 ng of wild-type plasmid DNA template, 5 fl of 10 x Tag Buffer, 1 pl of 10 mM each dNTP, 5-10 pl of NuSieve agarose from PCRl containing the megaprimer, 30 pmol of primer b (see Fig. 3). The reaction mix was overlaid with 50 p1 of mineral oil and subjected to the following regime: 95 "C for 4 min, during which 1 unit of Taq polymerase was added, then 95 "C for 1 min; ramp rate 6 SPC to 65 "C for 30 s; ramp rate 2 s/"C to 55 "C for 1 min; 72 "C for 1 min for 25 cycles, then 72 "C for 4 min and was held at 37 "C until required to prevent the gel setting.

The final PCR product was purified by diluting the PCR reaction mix 7-fold in phenol extraction buffer (300 m~ NaCI, 100 mM Tris-HC1, pH 7.0, I mM EDTA) and extracting with an equal volume of phenol/ chlorofodisoamyl alcohol (25:24:1) preequilibrated with phenol extraction buffer, then with CHClJisoamyl alcohol (24:l). Traces of agarose were removed by chilling on ice then centrifuging at 15,000 rpm (Sigma) prior to ethanol precipitation of the supernatant.

Construction of Mutant Plasmids Both the mutant PCR fragment and pGOFl DNA were digested with

suitable restriction enzymes; in early experiments PstI was used, then later, to restrict the length of PCR amplified DNA in the final construct, the enzymes NsiI and MluI were used. pGOFl was also treated with calf intestinal alkaline phosphatase (Sambrook et al., 1989). After re- covering the appropriate bands from a 2% NuSieve agarose gel, the fragments were ligated in a 50-pl reaction volume containing a 4:l ratio of insert (approximately 0.019 pmol) to vector (approximately 0.076 pmol). The gel slices were melted at 70 "C for 5 min and then trans- ferred to a 40 "C heat block before adding water, 10 x ligase buffer (Life Technologies, Inc.), and ATP all prewarmed to 40 "C. Ligase (1 unit) was added and the reaction incubated at room temperature for 1 h, then overnight at 15 "C.

Competent cells of E. coli DHl or XL-1 Blue were prepared by the CaCl, method and were transformed as described by Sambrook et al. (1989) except that ligation reactions containing agarose were heated to 70 "C for 5 min to melt the agarose. A 10-25-pl aliquot was then added to 100 pl40 mM CaCI,, 12.5 mM Tris-HC1, pH 8.0, a t 40 "C, mixed, and placed on ice for 5 min before the addition of 100 pl of competent cells. Mutant clones were confirmed by DNA sequence analysis.

Fungal lkansformation Culture conditions and transformation ofA. nidulans were according

to the procedures described by Ballance and Turner (1985) and Ballance et el. (1983). Stable transformants were isolated by repeated subcultur- ing of uracil-independent sporulating colonies.

Zndicator Plates for Detecting Galactose Oxidase-expressing lkansformants

Isolates expressing active galactose oxidase were identified by grow- ing on indicator plates ofAspergillus minimal agar medium (Rowlands and Turner, 1973) containing L-galactose to 2%, MgSO, to 2 m ~ , p - aminobenzoic acid to 1 pg/ml, ABTS to 1.2 mg/ml, and horseradish peroxidase to 0.04 unit (0.4 pg)/ml. Galactose oxidase-expressing transformants resulted in formation of a green coloration following incuba- tion for between 30 min and several hours at 30 "C.

Polyacrylamide Gel Electrophoresis (PAGE) of Galactose Oxidase Samples of purified galactose oxidase were routinely analyzed by

PAGE. In addition, transformants expressing inactive or low activity forms of galactose oxidase were screened by immunological analysis of SDS-PAGE Western blots. An aliquot of lo7 spores was used to inoculate

Structure and Mechanism of Galactose Oxidase 25097

50 ml of Aspergillus minimal medium (Rowlands and Turner, 1973) containing 16 CuSO, and was grown with aeration at 30 "C. After 4-6 days 10-ml aliquots of culture supernatant were removed with glass wool-plugged pipettes, buffered by the addition of Tris-HC1, pH 7.8, to 50 mM, then precipitated by the addition of (NH,),SO, to 80% saturation. After 30 min on ice, the precipitate was collected by centrifugation (25,000 x g) at 4 "C and the pellet resuspended in 0.1 ml of protease inhibitor buffer (1 ml of 50 mM Tris-HCI, pH 7.0, containing 10 pl 2% phenylmethylsulfonyl fluoride in propan-2-01 and 10 p1 of 0.35% tosyl- phenylalanine chloromethyl ketone in propan-2-01), The redissolved pel- lets were immediately fractionated through 10% polyacrylamide, 0.1% SDS gel (Hames, 1981). The markers used were 5 pl of low molecular weight marker proteins (Pharmacia Biotech. Inc.) for Coomassie- stained gels or 2.5 p1 of prestained markers (Bio-Rad) for gels to be Western blotted.

Anti-galactose Oxidase Antiserum Galactose oxidase denatured by heating to 100 "C for 5 min in 0.1%

SDS was used to raise antiserum in mice essentially as described by Harlow and Lane (1988). The first injection was prepared by sonication of 60 pg of galactose oxidase in phosphate-buffered saline (PBS) with an equal volume of Freund's complete adjuvant (Sigma), and the emulsion was administered by intraperitoneal injection into 6-week-old female BALB/c mice. Two further injections were administered at 4-week intervals and were comprised of 50 pg of galactose oxidase and Freund's incomplete adjuvant (Sigma). Serum was collected 2 weeks after the final injection, and control serum was obtained from age-matched un- treated mice.

Western Blotting Polyacrylamide gels were Western blotted according to Burnette

(1981) using an electroblot apparatus (Atto Corp., Tokyo). The filter was preincubated in 5% dried milk powder (Marvel) in PBS, washed (3 x for 10 min in PBS), then incubated for 1 h at room temperature in anti- galactose oxidase antiserum (1:5,000 dilution in 2 mg/ml bovine serum albumin, PBS, 0.02% Thimerosal). Antibody binding was then detected by washing the filters (3 x 10 min in PBS) before incubating for 1 h at room temperature with a secondary anti-mouse-IgG/peroxidase conju- gate (1:4,000 dilution). After further washes, galactose oxidase-specific bands were identified with chloronaphthol reagent. The reaction was stopped by rinsing the filter in water.

Purification and Assay of Galactose Oxidase

viously designated D. dendroides (NRRL No. 2903; Ogel et al., 1994), Galactose oxidase from the native organism, a Fusarium spp., pre-

was purified as described by McPherson et al. (1992b), which involves affinity purification on a column of Sepharose 6B, or by the modified purification procedure described below. Both procedures yield pure enzymes as judged by SDS-PAGE, although the modified procedure is more convenient and better suited to purification of active site mutants.

A. nidulans transformant cultures, inoculated with 5 x lo7 conidia, were grown in 20 x 1 liter (for pGOF1) or 5 x 1 liter (for pGOF101) of Aspergillus minimal medium containing 16 pa CuSO, at 30 "C with shaking (180 rpm). For pGOFl transformants, growth was allowed to proceed for 5 days. For pGOFlOl transformants, high level expression from the glaA promoter was induced by the addition of maltose to 1% (w/v) after 3 days, and growth was allowed to proceed for a further 3 days.

For pGOFl transformants, the culture medium was filtered successively through Whatman No. 1 filter paper, Whatman GFA grades D, B, and F to remove all traces of mycelium. The filtrate was concentrated to 2 liters on a Filtron pressure dialysis concentrator (Flow Laboratories), then diluted with an equal volume of cold distilled water and reconcen- trated twice with the final volume being reduced to 500 ml before overnight dialysis against 10 nm sodium phosphate buffer, pH 7.3. For pGOFlOl transformants, the smaller volume of culture medium was filtered through Whatman No. 1 filter paper, then ammonium sulfate was added to 30% saturation (w/v), and after centrifugation the supernatant was adjusted to 80% saturation (w/v) with ammonium sulfate. The pellet was dialyzed against 10 mM sodium phosphate buffer, pH 7.3.

Galactose oxidase was purified by chromatography through a column (2.5 x 25 cm; 125-cm3 bed volume) of cellulose phosphate, fibrous form (Sigma) in 10 II~M phosphate buffer, pH 7.3. After washing with 1 liter of 10 mM phosphate buffer, pH 7.3 (50 mlh), galactose oxidase was eluted with a linear gradient of 200 ml of 10 mM + 200 ml of 100 m phosphate buffer, pH 7.3 (25 ml/h); elution of protein was monitored by absorbance at 280 nm. Peak fractions were analyzed by SDS-PAGE, pooled, and concentrated in an Amicon ultrafiltration cell prior to storage in liquid

nitrogen; the enzyme for crystallization was used immediately without freezing.

Copper Saturation of Galactose Oxidase To ensure full copper occupancy, the galactose oxidase samples were

treated with Cu(NO,), at pH 6.1 to keep Cuz+ in solution, and then excess Cu2+ was removed by dialysis against pH 7.0 buffer because galactose oxidase has a lower aEnity for Cu" at lower pH values? The protocol was as follows. Enzyme was thawed from liquid nitrogen and dialyzed successively against 20 mM PIPES, pH 7.0; 20 mM PIPES, pH 6.1; 20 mM PIPES, pH 6.1, 1 mM Cu(NO,),; 20 mM PIPES, pH 7.0; 100 mM sodium phosphate buffer, pH 7.0, treated by passing through a column of chelating resin (iminodiacetic acid; Sigma) to reduce trace contamination with copper. All containers and pipettes used during removal of excess copper were acid washed and rinsed with distilled water that had been treated with the chelating resin. The galactose oxidase was centrifuged at 18,000 x g in a refrigerated microcentrifuge (Sigma) for 5 min to remove any insoluble material, then was concentrated in a Centricon-30 ultrafiltration device. The A,,, was measured before returning the protein to liquid nitrogen.

Atomic Absorption Spectrometry Copper analysis of aliquots of galactose oxidase was performed by Dr.

M. Hart (Micro Analytical Laboratory of the Department of Chemistry, University of Manchester) by optical emission spectroscopy (Perkin Elmer 6500XR ICP) using an inductively coupled plasma for the excitation source; the copper standards were SPEC PURE quality.

Measurement of Protein Concentration Galactose oxidase concentrations were determined by measuring

Azso. For native galactose oxidase, 1 absorbance unit corresponds to 0.65 mg/ml (Ettinger, 1974). The validity of this figure for the mutational variant proteins was tested by comparing protein concentrations of native, expressed wild-type and variants determined by A,,, measurements against the microbiuret method of Ithzaki and Gill (1964).

Ferricyanide Oxidation and Desalting Dialyzed galactose oxidase samples were thawed and incubated in

the presence of 0.05 M &Fe(CN), (potassium ferricyanide) for 16 h at 20 "C to oxidize the enzyme fully. Ferricyanide was removed by succes- sive cycles of concentration and dilution using a Centricon-30 ultrafiltration device until the A,,, of the filtrate was less than 0.001, corresponding to less than 0.5 PM &Fe(CN),, and giving an 80-270-fold molar excess of protein over ferricyanide.

Galactose Oxidase Assay and Kinetic Analysis A coupled assay system, based on the method of Amaral et al. (1963)

was used for qualitative and quantitative assays, including measurements of kinetic parameters. The reagent mix contained 8 mg of D-galactose, 20 mg of ABTS, and 3.3 mg (300 units) of horseradish peroxidase/ZO ml of 100 nm sodium phosphate buffer, pH 7.0.

For qualitative assays, 90 pl of reagent was placed in the well of a microtiter plate, 10 pl of protein solution was added, and the development of green coloration was observed. This test was also convenient for crude analysis of culture medium or even of mycelium.

For quantitative spectroscopic assays, 50 pl of protein solution was

ured at 25 "C. mixed with 1.0 ml of reagent, and the rate of change in A410 was meas-

Enzyme kinetic measurements with freshly ferricyanide-oxidized enzyme were performed over a range of D-galactose concentrations from 2.5 to 200 mM in a 3.0-ml reaction volume at 25.0 "C, verified with an electronic thermometer probe. The rate of change of A,,,, was recorded on a Pye-Unicam SP8-100 spectrophotometer and K,,, and V,, values calculated by using a nonlinear regression analysis program, ENZFIT, to fit the data to the Michaelis-Menten equation (Herries, 1984). The amounts of mutant protein required to determine measurable changes in absorbance were 60-100 times greater than for wild-type.

Visible Spectroscopy Solutions of fresh ferricyanide-oxidized protein, in 100 mM sodium

phosphate buffer, pH 7.0, within the concentration range 5-9 mg/ml, were studied over the region 900-300 nm on a Uvikon 860 W M S spectrophotometer (Kontron Instruments). A sample of the final filtrate from the Centricon unit, appropriately diluted, was used as the refer- ence so that absorbance contributed by any trace ferricyanide would be subtracted from the spectrum. Spectral data sampled at 1-nm intervals

P. F. Knowles, unpublished observations.


aB&c 10.5-kh i 6 4 9-kb Bo 6.5-kh B X

1 and Scal

Purify 3.2-kb fragment

Digest with Clal

C C ‘“4

J Ligate

R

1 Add &Ill linkers

9 \L ~ Digest with BglII ~ and Xbol

cx

$ E‘coRV Digest with

1’ I

Digest with &I11 and Xbal

FIG. 2. Cloning schemes for construction of plasmids for fungal expression of galactose oxidase. Relevant restriction sites are shown: B, BglII; C , ClaI; Sc, ScaI; X, XbaI. The galactose oxidase coding region is shown as a solid black band. Panel a , pGOF1, based on the vector pRG4, expression of the galactose oxidase gene is directed by the native promoter (A). Panel b, pGOF101, based on the vector pGPT-pyrG, in which the galactose oxidase coding region was cloned between the transcription terminator of the A. niger glucoamylase gene (heavily hatched band) and the promoter of A. awamori glucoamylase gene (lightly hatched band) which directs the maltose-inducible expression of galactose oxidase.

were down-loaded to an Atari ST 1040 for spectral processing. Evalua- tion of the data was made using the public domain program RSPLOT (R. J. Silliker, [email protected]).

Crystallization of Mutational Variant Proteins Sitting drop vapor diffusion microtiter plates, made from Linbro

tissue culture multiwell plates with microbridge inserts from Crystal Microsystems, were used in the crystallization. The plates were placed in a thermostatically controlled room at 18 “C. A. nidulans-expressed wild-type and mutant (W290H and C228G) proteins crystallized under the same conditions as the native Fusarium spp. galactose oxidase (It0 et al., 1991). The precipitant was ammonium sulfate in the concentration range 1.0-1.8 M. This was diluted from a 4.0 M stock solution using 0.8 M sodium acetate buffer in the pH range 4.1-5.2. The sitting drops consisted of 10 p1 of mutant protein solution a t 2.9 mg/ml to which 7 pl of well solution was added and allowed to mix without agitation. Crys- tals appeared within a few days. In the case of the C228G mutant, trials were also set up in which the protein solution had been dialyzed first against copper sulfate solution to ensure maximal occupancy of the copper site. The W290H mutant trials gave only twinned crystals, and streak-seeding (Stura and Wilson, 1991) was used to obtain x-ray quality single crystals. The C228G mutant trials, where the protein had not been dialyzed against copper sulfate solution, also produced only twinned crystals, but the trials using C228G mutant protein dialyzed against copper sulfate solution gave x-ray quality single crystals.

Data Collection and Structure Solution Diffraction data were collected on a Siemens-Nicolet XlOOA area

detector mounted on a Rigaku RU-200 rotating anode x-ray generator. The frame data were processed using XDS (Kabsch, 1988). Data reduction and all subsequent crystallographic computations were carried out by the CCP4 program suite (SERC Collaborative Computing Project no. 4 (1979); a suite of programs for protein crystallography, The Daresbury Laboratory, Warrington WA4 4AD, U. K.). The mutant protein crystals were essentially isomorphous with the native Fusarium spp. galactose oxidase crystals (space group P2,, a = 98.0 A, b = 89.4 A, c = 86.7 A, b = 117.8”). Focmutanu - Foo(native) and Focmutant, - Fccnative, difference maps were studied on an Evans and Sutherland PS300 graphics system using the program FRODO (Jones, 1978). The molecular model refinement was by the Hendrickson-Konnert least squares method.

RESULTS

Heterologous Expression of Galactose Oxidase-% allow protein engineering studies of an enzyme, it is necessary to express the mutant proteins in a host background that does not express the same or a similar enzyme. For galactose oxidase expression we selected A. nidulans as a suitable host since ( a ) it is a well characterized filamentous fungus for which appropriate ge- netic manipulation systems are available; ( b ) it is a proven host for the high level expression of heterologous genes and the production of secreted proteins; and ( c ) it does not contain endogenous galactose oxidase activity.

pGOFl was generated as shown in Fig. 2a by subcloning a 3.14-kb ClaI fragment from pGAO9 into the fungal vector pRG4 so that expression of the gaoA gene is regulated by its own promoter. The 3.14-kb fragment comprises 851 bp of untranslated upstream sequence, including 13 bp of pBluescript polylinker sequence, the 2,040-bp galactose oxidase coding region and 263 bp of untranslated downstream sequence. Al- though there is another ClaI recognition sequence (ATCGAT) within the subcloned upstream region (nucleotides +60 to +65; McPherson et al., 1992b) this is followed by a C and was not cleaved by ClaI, probably because of the E. coli dam-directed methylation system which recognizes and methylates the sequence GATC (Messer and Noyenveidner, 1988). The gaoA region in pGOFl was sequenced and proved to be identical to the sequence determined previously for pGAO9 (McPherson et al., 1992b).

pGOFl was used for the construction and expression of the mutational variants investigated in the present study; however, the yields of protein were relatively low (2-5 mgAiter) so an improved system, pGOF101, was developed and used for the isolation of wild-type expressed enzyme during the present study. In pGOFlOl the gaoA coding region was brought under the control of the inducible and highly expressing A. awamori glaA (glucoamylase) promoter as shown in Fig. 2b. pGAOll


was digested with EcoRV which cleaves 84 bp before the initi- ating ATG codon of the galactose oxidase coding region; this site was modified by the addition of BglII linkers. The gaoA coding region contains a single BglII site so that subsequent digestion with BgZII and XbaI released two fragments of 1.1 kb and 1.6 kb, which were ligated with XbaIIBgZII-digested pGPT-pyrG to generate pGOF101, which contains a 2,710-bp insert. The DNA sequence of this insert was determined and proved to be identical to the corresponding region of pGAO9 (McPherson et al., 1992b).

Comparison of the Expression Systems-In the present study, wild-type enzyme was produced from the original Fusarium spp. NRRL 2903 strain (native wild-type enzyme) and from A. nidulans transformed with both pGOFl and pGOFlOl (expressed wild-type enzyme). The mutant proteins were all produced from pGOFl transformants, prior to the development of pGOF101. Transformation of A. nidulans with pGOFl or pGOFlOl resulted in one to seven mitotically stable transformants per pg of DNA. However, the integration patterns, gene copy number, and levels of enzyme produced by the pGOFl and pGOFlOl transformants differed significantly.

Typically pGOFl yielded 2-5 mg of galactose oxidaseAiter, which is comparable with levels obtained from Fusarium spp. NRRL 2903. However, hybridization analysis of Southern blots of genomic DNA from pGOFl transformants (data not shown) revealed a complex hybridization pattern suggestive of multi- ple integration events at several sites within the genome, and usually 10 or more copies of the gene. It usually proved necessary to screen up to 30 transformants to identify one with a suitable level of galactose oxidase expression and with no unusual growth characteristics (such as poor growth rate or excess polysaccharide production) that prevented normal isolation of enzyme. By contrast, pGOFlOl transformants, induced with maltose, yielded typically 30-50 mg of galactose oxidase/ liter, and in some cases levels were even higher. Hybridization studies have revealed a single integration site at which there was one or more copy of the gaoA gene. There were no unusual growth characteristics of pGOFlOl transformants.

The difference in integration characteristics of the plasmids probably results from heterologous (pGOF1) and homologous recombination (pGOF101) events. Although thepyr4 gene product of N. crassa and the pyrG gene product of A. nidulans are functionally equivalent, there is little DNA sequence homology between the two genes; therefore the pyr4 gene of pGOFl will not preferentially recombine with thepyrG gene ofA. nidulans. In the case of pGOF101, homologous recombination between the pyrG genes of the plasmid and chromosome would lead to site-specific integration of the gaoA gene at the pyrG locus, and there would be no disruption of other genes within the genome. However, with pGOF1, integration probably occurs by heterologous recombination, at several distinct positions in the genome. It seems likely, given the range of morphological and physiological variation observed amongst pGOFl transformants, that integration events frequently lead to gene disruption, which is an undesirable feature of the system. pGOF101, therefore, provides a good expression system with predictable integration characteristics and high level, inducible expression of the gaoA gene.

Preparation of Mutational Variants by PCB-The 3 residues of specific interest during the present study are TY?~, CysZz8, and TrpzS0. Tyr2I2, an equatorial copper ligand that is involved in the thioether bond and is the probable site ofthe free radical, was altered to phenylalanine W272F) to remove the phenolate 0. CysZz8, the other residue involved in the thioether bond, was replaced by glycine (C228G), which removes both the Cp and Sy. The side chains of Tyr272 and CysZz8 form an extended aro-

A. PCR 1

+ a

4-

v-“

B. PCR2 h R1 R2

R I + C. Restriction digest R I J. IR2 Y

D. Ligation + R l RI R2 R2 - v

J. E. Mutant plasmid

FIG. 3. Schematic of the PCR mutagenesis strategy. Arrows

PCRl and PCR2 respectively; m, mutagenic primer. R l and R2 repre- show the orientations of the primers; a and b, flanking primers used in

sent restriction sites. The strategy involves several stages; PCRl gen- erates a megaprimer that is incorporated into a larger fragment during

is ligated with similarly digested plasmid to regenerate a galactose PCRS; this fragment is digested with suitable restriction enzymes and

oxidase gene containing the desired mutatiods).

matic plane that is suitably located for a stacking interaction with TrpzgO which was altered to histidine (W290H) to retain a heterocyclic ring.

APCR-based method, schematically illustrated in Fig. 3, was used to generate mutations. This procedure is based on the gene splicing by overlap extension approach (Horton and Pease, 1991) involving the use of a PCR-generated mutant “megaprimer” in a second PCR (Sarkar and Sommers, 1990) to produce a specifically mutated fragment of the target gene which is then used to replace the corresponding region of the wild-type target gene. This mutagenesis procedure generally yielded 80-90% mutants among full-size clones which were therefore screened by single dideoxynucleotide chain termina- tion reactions to identify mutants. The entire gaoA gene was then sequenced to ensure the presence of the desired mutation(s) and the absence of unselected mutations. We have found sequencing of the entire coding region to be a critcal step in avoiding two sources of error. First, and most common, was the nontemplate-directed addition of a base, usually adenine, to the 3’-ends of the product from PCR1. Despite the concept that the 3’-end of a primer must be perfectly matched to the template for successful priming during PCR, we have found that the presence of a 3’-mismatch (AC, AG, or A:A) between the megaprimer and template does not prevent specific priming and therefore leads to fixation of unselected mutations. In later experiments we overcame this problem, as proposed by Kuipers et al. (1991), by designing PCR primers whose 5’-end was ad- jacent to a thymine. Second, we identified two examples of unselected coding sequence mutations outside the PCR-amplified region. This implies that mutations can arise at a low but significant level during in vitro manipulations of DNA andor in

25100 Structure and Mechanism of Galactose Oxidase TABLE I

Galactose oxidase purification

expressing either wild-type or mutant forms (W29OH or C228G) forms of galactose oxidase. The purification scheme involved concentration of the Protein was prepared from the culture supernatants of Fusarium spp. NRRL 2903 (native wild-type enzyme), and A. nidulans transformants

supernatant followed by phosphocellulose chromatography.

Enzyme and procedure Volume Concentration Total units Protein Total protein Specific activity Yield

Native (Fusarium spp.) Initial culture superna-

Phosphocellulose chro-

Wild-type (Aspergillus ex-

Initial culture superna-

Phosphocellulose chro-

tant

matography

pressed)

tant

matography W290H


Phosphocellulose chro- tant

C228G matography


Phosphocellulose chro- tant

matography

ml

25,000

25

4,000

43

20,000

13

10,000

18.5

unitslml

6.37

4121

80.03

3616

ND

2.52

ND

0.115

159,000

103,000

320,100

155,500

ND

32.8

ND

2.13

mglml

ND“

3.68

ND

3.31

0.004

4.64

0.01

3.28

mg

ND

92

ND

142.3

80

60

100

61

unitslmg %

ND 100

1120 63.5

ND 100

1093 48.6

ND 100

0.54 75

ND 100

0.035 61

” ND, not determined.

vivo replication of plasmid. This observation has serious impli- cations for some protein engineering experiments since it chal- lenges the view that it is sufficient to sequence only PCR- derived regions that are inserted within a non-PCR-amplified gene backbone. It is clearly important to sequence the complete coding region of every new mutant construct irrespective of the origin of the component DNA segments.

Purification of Galactose Oxidase and Mutational Variants-We have developed a simplified and rapid procedure for purification of galactose oxidase based on a single chromato- graphic separation using cellulose phosphate. The protocol yielded 60-90 mg of galactose oxidase from 20 liters (pGOF1 transformants) and 140 mg of galactose oxidase from 4 liters (pGOF101 transformant) of culture supernatant. Native and expressed wild-types and the mutant proteins, W290H and C228G, behaved in a similar manner during the purification process, and Table I summarizes the purification data for these enzymes. The only problem encountered was the slightly lower than expected yield of expressed wild-type from the pGOFlOl transformant where it seems that the higher concentration of protein led to greater than expected losses a t each step because of the limited solubility of the protein and consequent precipitation.

Purification of native and expressed wild-type forms of the enzyme was monitored by assaying protein-containing fractions for galactose oxidase activity. Mutant protein samples with low activity were analyzed by Western blot analysis of SDS-polyacrylamide gels.

Despite several attempts using a variety of independent transformants, i t proved impossible to isolate intact Y272F protein. Western blot analysis indicated the presence of several smaller species consistent with degradation products, despite the inclusion of proteinase inhibitors during protein isolation (see “Experimental Procedures”). In this variant, one of the important equatorial copper ligands ( T y P 2 ) has been removed so this protein may not be capable of binding copper. I t is known that the presence of copper is important in maintaining the stability of the enzyme (Kosman et al., 1974), and it is difficult to purify a copper-free form of galactose ~ x i d a s e . ~

Protein Concentrations-An important issue for comparative studies of variant proteins is accurate estimation of protein

a b c d e f

94

67

43

FIG. 4. Western blot analysis of 10.6% polyacrylamide-SDS gel of galactose oxidase proteins. Lune a, C228G, lane b, native wild- type; lane c, expressed wild-type; lane d, C228G; lane e, W290H; lane f, molecular size markers, values are indicated in kDa. The C228G protein, which lacks the thioether bond, migrates a t a slower rate than the other species.

concentrations. A,,, measurements are influenced most significantly by tryptophan residues, so estimates of concentration of the W290H variant,, which has 15 rather than 16 tryptaph-an residues, were expected to differ from those of the other proteins. The ratios of the values obtained from the microbiuret assay (Ithzaki and Gill, 1964) and A,,, measurement (means 2 standard error for at least six experiments for each protein) were 0.96 2 0.01 for wild-type forms, which is in agreement with Ettinger (1974), 1.015 2 0.03 for C228G and 1.048 2 0.01 for W290H. The ratio for the W290H variant is significantly higher (at the 99% confidence limit) than the wild-type ratio, by a factor of 1.09, which is consistent with the prediction of a factor of 1.067 from the tryptophan content of wild-type galactose oxidase (Trp = 16) and W290H ( T r p = 15). Protein concentrations of W290H derived from A,,, measurements were therefore corrected by multiplying by 16/15.

Polyacrylamide Gel Analysis of Proteins-Fig. 4 shows a Western blot of an SDS-PAGE of the various purified proteins. The mobility of native wild-type, expressed wild-type, and W290H variant were identical with an apparent size of 65 kDa. By contrast the C228G variant migrates as an apparently larger species of 68 kDa. The estimated size of the C228G

Structure and Mechanism of Galactose Oxidase

- m 0 L '

I \ \

25101

FIG. 5. Comparison of the refined models of the Fusarium spp. native galactose oxidase and the Aspergillus expressed mutant proteins showing the changes around the active site. Blue represents positive density, and red represents negative density; the wild-type structure is shown in orange. Panel a , comparison of the native with W290H the 2.1 A F, - F, difference map is contoured at the 3.5~ level. Panel 6, comparison of native with C228G the 2.6 A Fo - F, difference map is contoured at the 3.5 u level.

mutant is in good agreement with the size of 68.5 kDa for the the presence of the thioether bond in native, expressed wild- mature enzyme calculated from DNA sequence data (McPher- type, and W290H forms, would cause the 44 residues between son et al., 1992b). The change in mobility correlates with the CysZz8 and T ~ I ? ~ ~ to form a peptide loop leading to a less ex- presence of the thioether bond. In the C228G variant the ab- tended conformation of the polypeptide chain resulting in a sence of this bond will result in a fully extended, linear confor- faster migration rate during SDS-PAGE. mation of the galactose oxidase polypeptide chain giving an Crystal Structure of the Aspergillus-expressed Wild-type accurate estimate of molecular size by SDS-PAGE. By contrast, Protein-The x-ray diffraction data were usable to 2.0Awith an


- His290

%"-.-, Q Ace705

FIG. 6. The additional acetate binding site in the AspergiZZus expressed W290H mutant protein. Residues 326-328 are from a symmetry-related molecule. Hydrogen bonds are indicated by dashed lines.

R,, on intensities of 5.4%. Difference maps indicated that there was essentially no difference between this crystal structure and that of the native, Fusarium spp. galactose oxidase, pH 4.5. However this structure and both mutant structure difference maps revealed a region of positive electron density between domain 1 of one molecule and domain 2 of a symmetry- related molecule. This appears to be because of a bound sulfate ion from the crystallization mother liquor. This was not observed in the original Fusarium spp. galactose oxidase structure (It0 et al., 1991) as the mother liquor had been changed to facilitate preparation of heavy atom derivatives and no longer contained sulfate ions. Two of the sulfate oxygens are hydrogen bonded to the protein; one to the headgroup of ArglZ2 and the other to the side chain hydroxyl of T h P 8 from a symmetry- related molecule. After refinement the final R factor was 17.1% for all reflections between 10 and 2.0 A. Geometry is good with a root mean square deviation from ideal bond length of 0.014 A.

Crystal Structure of the W290H Mutant Galactose Oxidase- The diffraction data were usable to 2.1 a with an R,, on intensities of 4.6%. The difference maps indicated only local changes around the mutation site (Fig. 5a). After minor re- building and refinement, the final R factor was 15.7% for all reflections between 10 and 2.1 A, and the root mean square deviation from ideal bond length was 0.017 8. In the mutant structure the Hiszg0 ring is oriented and positioned in the same manner as the five-membered ring of the wild-type galactose oxidase TrpZg0. In the native wild-type enzyme the side chain of Sel.291 is in a g- conformation, pointing away from T r p Z g 0 ,

whereas in the mutant protein, it adopts a g+ conformation, pointing into the gap created by the substituted residue. This conformation is not possible in the wild-type protein because of a steric clash with the T r p Z g 0 side chain. An ordered water molecule hydrogen bonded to the imidazole nitrogen of Hiszw helps to fill the space created by the substitution. It has been proposed that !I'rpZg0 might act as the general base during catalysis (Ito et ul., 1992) since the abstracted pro-S proton of the substrate is oriented directly toward the ring NE of T r p z g O in modeling experiments with native enzyme. It was anticipated that the NE of Hiszgo would adopt a similar position. However, the presence of the ordered water molecule is consistent with the Hiszgo ring spending some of the time in the flipped conformation in which NE of Hiszgo coincides with the position of the CE of WZg0 in the native enzyme (Fig. 5a).

Interestingly, an additional acetate ion is found several ang- stroms from the mutation site (Fig. 6). The protein structure around this ion appears to be little changed compared with the native protein. The ordered water structure has changed to accommodate the acetate ion, with two waters in the wild-type being replaced by a single water hydrogen bonded to one of the acetate oxygens. The ordered water that is hydrogen bonded to

the Hiszg0 imidazole nitrogen is in Van der Waals contact with this acetate oxygen. The other acetate oxygen forms a bifur- cated hydrogen bond to the main chain amides of Glylg6 and

from a symmetry-related molecule. The position of the acetate ion does not sterically clash with the position of the wild-type tryptophan, so its presence appears to result from the altered network of ordered waters in the mutant protein crystal structure.

The structure of the copper binding site displays no significant differences from the native galactose oxidase structure.

Crystal Structure of the C228G Mutant Galactose Oxidase- The crystals of C228G did not diffract as well as wild-type, and good data were obtained only to 2.6 A with an R,, on intensities of 7.0%. The final R factor for the model was 14.9% on all reflections between 10 and 2.6 A, and the root mean square deviation from ideal bond length was 0.020 A. The crystal structure reveals no change in the position of the main chain or the copper and its ligands. This includes w7', which is co- valently bonded, in the wild-type protein, to via a thioether linkage. However, the side chains of TrpZg0 and PheZz7 have moved toward the space created by the loss of the cysteine (Fig. 5b). The side chain of Phelg4 has also moved to occupy the space created by the displacement of Phezz7.

Visible Spectroscopic Studies on Native and Mutant Forms of Galactose Oxidase-Fresh samples of ferricyanide-oxidized enzymes were used for spectroscopic experiments. The use of Centricon filtration units allowed rapid removal of ferricyanide (within 3 h) and gave good recovery of protein (between 85 and 95%). The protein that was lost appears to have precipitated and was removed by centrifugation after each Centricon filtration step. The C228G and W290H proteins appeared to be more prone to denaturation than the native or expressed wild-type forms, indicating that these variant forms are less stable.

Examples of the visible spectra of native, expressed wild- type, W290H, and C228G forms of galactose oxidase in their activated states are shown in Fig. 7. Copper analyses, shown in Table 11, indicate that the native and expressed wild-type proteins have approximately 1 mol of copper/mol of protein, the W290H variant has 1.5 mol of copper/mol of enzyme, whereas the C228G mutant has only 0.25 mol of copper/mol of protein.

Spectra from the native and expressed wild-type proteins (Fig. 7) are broadly similar and are consistent with the data from previous studies (Whittaker and Whittaker, 1988). The

values are 5060 for native enzyme and 5230 for the wild- type expressed. The two mutant proteins show differences in both the wavelength and magnitude of the absorbance peaks. For W290H, the broad peak at 810 nm is absent, and the peak in the 400-500 nm region has shifted from 445 to 476 nm, with an extinction coefficient of 2,800 M - ~ cm". The C228G variant also lacks the 810 nm peak and has a very small 476 nm peak, E = 500 M - ~ cm", which is likely to be due in part to the low copper occupancy of only 0.25 mollmol protein.

The spectra for both mutant proteins show no response to ferricyanide treatment, although the W290H variant does show a 1.6-fold higher activity following treatment. The origin of this effect requires further study. It is interesting that the level of additional copper shows a close correlation with the increase in activity following the ferricyanide treatment which includes several washing steps that could potentially remove loosely bound copper. By contrast, as shown in Fig. 8, the wild-type forms show a 6-fold increase in esl0 and 4-fold increase in E ~ ~ ~ ,

and the new spectrum of the wild-type enzyme persists for several days suggesting the oxidized state of the enzyme is stable.

Enzyme Kinetics-Enzyme kinetic measurements were made for native wild-type, expressed wild-type, W290H, and C228G


300 350 400 450 500 550 600 650 700 750 800 850 900

Wavelength (nm)

from Aspergillus; ii, native wild-type enzyme from Fusarium spp. NRRL 2903; iii, W290H variant enzyme; iu, C228G variant enzyme; iu(a), C228G FIG. 7. Visible spectrum of wild-type (ferricyanide-oxidized) and mutant forms of galactose oxidase. i, expressed wild-type enzyme

variant enzyme shown on an expanded scale. For details of sample preparation see "Experimental Procedures."

TABLE I1 Copper content of wild-type and mutant forms of galactose oxidase Excess copper was removed from samples by filtration in Centricon

filter units. Protein copper contents were corrected for levels of copper remaining in the final Centricon filtrates.

Copper Protein concentration copper/

Concentration Final Centricon protein filtrate ''ohin

W PM mol lmol Native wild-type 140 4.7 164 1.2

Expressed wild-type 143 1.6 134 0.9

W290H C228G

150 1.6 117

219 1.5 0.8 27.5 0.24

(Fusarium spp.)

(A. nidulans)

proteins, over a range of D-galactose concentrations from 2.5 to 200 m ~ . The results are summarized in Table 111, which includes k,, estimates to provide an indication of the catalytic efficiency of each enzyme. The data obtained generally produced a good fit to the Michaelis-Menten equation in agreement with the studies of Kwiatkowski et al. (1981), who showed that the galactose oxidase reaction followed this kinetic model. The plots of change in A420 against time with C228G were curved, and estimates of maximal rate at zero time were obtained by extrapolating to zero time and measuring the slope of the tangent to the curve at that point, which probably accounts for the slightly higher estimates for K, for this variant. The nonlinearity of these plots could be due to the large amount of enzyme required to give a measurable slope so that the propor- tion of substrate complexed with enzyme may no longer be regarded as insignificant, particularly with respect to oxygen. The concentration of C228G in the assays was 0.44 p ~ , and in the air-saturated solutions used for the measurements the oxygen concentration was about 290 p ~ , which only represents a 660-fold excess.

The plots with W290H were linear, although, interestingly at low substrate concentrations ( 4 0 0 m ~ ) or with enzyme that had not been ferricyanide treated, an increase in velocity was observed during the 1st min or so before reaching a stable maximal rate. This profile suggests an activation phenomenon, and it seems likely that the peroxidase present in the coupled assay system oxidizes the mutant galactose oxidase to a more active form.

DISCUSSION

Enzyme Expression and Purification-We have developed an efficient heterologous expression system for galactose oxidase by exploiting A. nidulans which does not produce an endogenous galactose oxidase. Plasmid pGOF101, in which the galactose oxidase coding sequence is expressed from an inducible, high level glucoamylase promoter, provides a convenient and reliable system for production of large amounts of protein. We have also simplified the purification scheme for galactose oxidase to a concentration process then a single phosphocellulose chromatography step. The purification scheme allows the rapid isolation of sufficient enzyme (approximately 40 mgkter) for a range of structural and enzymological studies.

Native and Expressed Wild-type Forms-The crystal structures of the native wild-type enzyme (It0 et al., 1991,1994) and the A. nidulans expressed wild-type (this study) were essentially identical. The spectral properties for both of these wild- type forms are similar to those reported by Whittaker and Whittaker (1988). The activated enzyme has absorbance peaks at 445 and 810 nm with extinction coefficients of approximately 5,100 M - ~ cm" and 3,260 M - ~ cm", respectively. The 445 nm band has been analyzed by Whittaker et al. (1989) as being five times more intense than would be expected for a tyrosine +

Cu(I1) charge transfer transition, and it was concluded that it is due to an overlapping transition involving the axial Tyr495 +

Cu(1I) charge transfer transition and the v + 7ic ring transition of the aromatic radical chromophore. The 810 nm band has been interpreted by Whittaker and co-workers (Whittaker et al., 1989; Whittaker and Whittaker, 1993) as resulting from a mixture of tyrosine + Cu(I1) charge transfer and charge resonance excitation between the aromatic v systems involved in the charge transfer complex. Whittaker and Whittaker (1993) comment that stacking of TrpZg0 over the " y P 2 4 CysZz8 thioether bond as revealed by the crystal structural studies (Ito et al., 1991) support this interpretation, and they speculate that the combination of the modified tyrosine and the stacked tryptophan most likely compose the radical site probed in the electronic spectra.

Y272F, W29OH, and C228G Variants-We were unable to purify any intact Y272F variant protein. The modified tyrosine residue, which is an equatorial ligand to the copper, is clearly of importance for copper binding, and it seems probable that the mutant protein is inherently less stable because of a deficiency


ia Wild-type 7000

6000

5000

4000

3000

2000

1000

1 I\\ a

C

300 350 400 450 500 550 600 650 700 750 800 850 900

Wavelength (nm) FIG. 8. Visible spectrum of wild-type enzyme from Fusarium spp. NRRL 2903 (native enzyme) and Aspergillus expressed wild-type

Fusarium spp. NRRL 2903; c, Fusarium spp. form before activation; d , Aspergillus expressed form before activation. enzyme, before and after activation by potassium ferricyanide. a , expressed wild-type enzyme from Aspergillus; b , native wild-type enzyme from

TABLE I11 Summary of kinetic parameters for wild-type and mutant forms of galactose oxidase

Eo represents the amount of enzyme present in each reaction and assumes an M, of 68,000.

Enzyme K for D-gaTrIctose E. kcat kc,@"!

m M pmol s-1 s-1 MI

Native wild-type (Fusarium spp.) 67.0 f 5.4 1.23 x 1O"j 2,990 f 110 44.6 k 5.4 to

7.36 x

to 9.37 x 10-6

Expressed wild-type (A. nidulans) 71.1 f 7.7 1.59 x 3,420 f 100 48.1 f 4.5

W290H 65.7 f 4.9 4.5 X 10-4 2.456 0.067 0.040 * 0.004 C228G 84.1 * 7.5 1.32 x 10-3 0.795 * 0.035 0.0095 * 0.0013

to 1.32 X 10-3

in copper binding. The W290H and C228G protein crystal structures confirm

the presence of the expected amino acid substitutions within the active site and reveal only minor local changes in the positions of certain amino acid side groups in the vicinity of the substitutions, relative to the wild-type structure. In the C228G mutant the thioether linkage is absent, and this correlates with a difference in mobility of the protein through SDS-PAGE compared with the wild-type and W290H proteins. This mobility shift is likely to prove useful as an assay for monitoring the formation of the thioether bond.

Both mutant proteins showed dramatic changes in spectral properties relative to wild-type galactose oxidase. The band at 445 nm is shifted to 476 nm, and the extinction coefficient is decreased to 2,800 M" cm" (W290H) and 500 M - ~ cm" (C228G), whereas the 810 nm band has been eliminated. The absorption band in the 400-500 nm region may be assigned to a combination of a ligand-to-metal charge transfer of the axial Tyr4g5 +

Cu(I1) and T + T* ring transitions of the equatorial w72 . The shift in band position from 445 nm (wild-type) to 476 nm (for variants W290H and C228G) suggests that the energy of the T + ?ic transition is influenced by the stacking tryptophan and the thioether bond. Since the axial Tyr495 3 Cu(I1) distance is not affected in either variant, the energy of this transition is expected to be perturbed only slightly. The decreased extinction coefficients for the two mutants at 476 nm indicate lowered levels of the tyrosine radical. Ferricyanide oxidation does not

significantly increase the amount of the tyrosine radical present, as judged by the 476 nm absorption peak, suggesting that the reduction potential of the tyrosine radical in the variants might have been increased relative to the wild-type. However there is some conflict between the spectral properties and the kinetic results, since the W290H variant does display a modest increase in activity (1.6-fold) following ferricyanide oxidation, which does not correlate with any change in spectral characteristics. It seems probable that this increase in activity is caused by the removal of inhibitory loosely bound copper during the ferricyanide treatment.

The shoulder at 625 nm evident in wild-type and variant forms may be assigned to Tyr' + Cu(I1) ligand-to-metal charge transfer. Whittaker et al. (1989) have shown that resonance Raman spectra of a modified tyrosine are observed with 659 nm excitation, which would fall within the absorption envelope of the 625 nm shoulder. Tyrosine ring modes are resonance en- hanced by excitation of ligand-to-metal charge transfer.

The absence of the 810 nm band in the W290H variant con- flicts with the suggestion that this band is due to a mixture of ligand-to-metal charge transfer and aromatic charge transfer (Whittaker et aZ., 1989; Whittaker and Whittaker, 1993). Since the 476 nm band in the W290H variant is relatively intense, this indicates the presence of a substantial amount of Tyr'. If a Tyr. 3 Cu(I1) ligand-to-metal charge transfer contributed to the 810 nm band then such a band should also be observed in this variant profile. This observation is consistent with assign-


ment of the 810 nm band as charge transfer in the stacked Tyr272-Trp290 unit and is the first direct evidence for this inter- valence transition; EPR and ENDOR studies (Whittaker and Whittaker, 1990; Babcock et al., 1992) support the involvement of the thioether modified tyrosine ( ! I ? y P 2 ) in the aromatic radical site but to date have provided no evidence for the further involvement of TrpZg0 in this site.

The very low extinction coefficient at 476 nm for C228G probably reflects the low copper content (0.25 Cdprotein). This mutant appears to have a decreased affinity for copper compared with the wild-type and W290H mutant forms. The origin of this effect is unclear at present but suggests that through the thioether bond, CysZz8 influences the ability of Ty?72 to act as a copper ligand. The very low measurable data for the C228G variant make comparisons between proteins difficult. Nonethe- less, the identical wavelength maximum (476 nm) in the visible spectrum of the W290H and C228G proteins, taken together with the approximately 4-5-fold difference in both extinction coefficient at 476 nm and kcaJKm values, which correlate re- markably well with the 4-5-fold lower copper occupancy of the C228G variant, tend to suggest that T r p Z g 0 and Cys228 both influence the free radical site of galactose oxidase in a similar manner, with CysZz8 additionally contributing to copper binding. On the basis of this similarity one might argue that TrpZg0 may not be involved in a specific catalytic event (Ito et al., 1992) since such a role would be expected to lead to a more significant difference between the mutant proteins than is observed; however, further experiments are required to address this question in more detail.

The studies reported in the present paper represent only a beginning to the characterization of the W290H and C228G mutants which require further investigation particularly by spectroscopic methods. It is clear, however, that together with the analysis of these and further mutational variants, including those incorporating unnatural amino acid residues (see for example, Judice et al., 19931, will help in understanding the structural requirements for the radical site in galactose oxidase.

Acknowledgments-We thank M. Reynolds for providing data from a galactose oxidase purification, Dr. A. MacGregor for raising the anti- galactose oxidase antiserum, and Dr. M. Hart for performing the copper

G. May, and Dr. M. Ward for providing strains and plasmids. analysis experiments. We are grateful to Professor G. Turner, Professor

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THE JOURNAL OF BIOL~~ICAL No. 40, 7, pp. 1994 by The … · 2001-06-29 · Structure and Mechanism...

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