Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli Viviana Job, Gianluca Molla, Mirella S. Pilone and Loredano Pollegioni Department of Structural and Functional Biology, University of Insubria, Varese, Italy We have cloned the gene coding for t he Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes gly- cine and sarcosine to the corresponding a-keto a cid, ammonia a nd hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21( DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-b- D -galactoside induction conditions, soluble and active chimeric GO was e xpressed up to 1.14 U g )1 of cell (and a fermentation yield of 3.82 UÆL )1 of fermentation broth). A n N-terminal His-tagged protein (HisGO) w as also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents  3.9% of the total soluble protein content o f t he cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 UÆmg )1 protein at 2 5 °C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of  180 kDa with the spectral properties typical of flavoproteins. G O exhibits good thermal s tability, with a T m of 46 °C a fter 30 min incubation; its stability is maximal in the 7.0–8.5 pH range. A comparison of amino- acid sequence and substrate specificity indicates t hat GO h as similarities to other flavoenzymes acting on primary amines and on D -amino acids. Keywords: g lycine oxidase; flavoprotein; oxidase; His-tagged protein; purification. Glycine oxidase (GO) is a flavoenzyme that was recently discovered followi ng the complete sequencing of the Bacillus subtilis genome [1]. A preliminary i nvestigation reported o n the e xpression o f the yjbR gene product and on its partial purification and characterization in Escherichia coli [2]. GO was found to be active on various amines such as sarcosine, N-ethylglycine, glycine and D -amino acids, and to partially share substrate specificity with both D -amino-acid oxidase (DAAO, EC 1.4.3.3) and sarcosine oxidase (SOX, EC 1.5.3.1) [2]. GO ca taly zes the o xidative de amination o f primary and secondary amines to give a-keto acids, ammonia and hydrogen peroxide (L. Pollegioni, unpub- lished r esults). SOX catalyses the o xidative demethylati on of sarcosine (N-methylglycine) to form glycine and formalde- hyde. Similarly, DAAO catalyses the oxidative d eamination of neutral and (with a lower efficiency) basic D -amino acids to give the corresponding a-keto acids and ammonia. In both cases, the reduced coenzyme is re-oxidized by molecular oxygen to yield H 2 O 2 . Although DAAO and SOX s how a wide s ubstrate t olerance, they do not efficiently oxidize glycine. The present aim of this project is to elucidate the structure–function relationships in GO, with the ultimate goal of clarifying the modulation of the substrate specificity in enzymes (GO, DAAO and SOX) that catalyse reactions on similar compounds. Although extensive information on the functional and structural characteristics of DAAO and SOX is available, there is little knowledge of GO p roperties and reactions. There is also a biotechnological aspect to this project, as the stereoselective reaction catalysed by DAAO is of considerable importance in biotechnology and indus- try, e.g. the bioconversion of cephalosporin C to glutaryl- 7-amino cephalosporanic acid [3,4]. Industrial interest i n t he discovery of amino-acid oxidase activities with new, wider substrate specificity is increasing. Because of the relevance of this topic, we cloned the B. subtilis DNA that encodes GO and studied the overex- pression of this enzyme in E. coli. I n t his p aper we report on two expression systems f or obtaining fairlylarge quantities o f a r ecombinant chimeric G O from E. coli cells and a quicker, high-yield procedure for the p u rification of this flavoenzyme. In addition, the physical and chemical characteristics, the activity on glycine and sarcosine, and the substrate specifi- city of the purified enzyme have been characterized. MATERIALS AND METHODS Materials Restriction enzymes and T 4 DNA ligase were o btained from Roche Diagnostics. Ampicillin, chloramphenicol, Luria–Bertani broth, o-dianisidine and horseradish peroxi- dase were purchased from Sigma. DEAE-Sepharose Fast Flow, phenyl-Sepharose CL-4B, Superdex 200, HiTrap Che- lating and PD10 prepacked columns were from Amersham Correspondence t o L. Pollegioni, Dipartimento di Biologia Strutturale e Funzionale, Universita ` degli Studi dell’Insubria via J.H. Dunant 3 , 21100 Varese, Italy. Fax: + 332 421500, Tel.: + 332 421506, E-mail: loredano.pollegioni@uninsubria.it Abbreviations: GO, glycine oxidase; TSOX, heterotetrameric sarcosine oxidase; MSOX, monomeric sarcosine oxidase; DAAO, D -amino-acid oxidase; DASPO, D -aspartate oxidase; DMGDH, dimethylglycine dehydrogenase; SDH, sarcosine dehydrogenase; PIPOX, pipecolate oxidase; INT, iodonitrotetrazolium chloride. (Received 23 O ctober 2001, revised 7 January 2002, accepted 16 January 2002) Eur. J. Biochem. 269, 1456–1463 (2002) Ó FEBS 2002 Pharmacia Biotech and hydroxyapatite was from Bio-Gel- HTP. All purification steps were p erformed using a n A ¨ KTA-FPLC system (Amersham Pharmacia Biotech). The plasmid DNA was extracte d and purified from E. coli cells using the FlexiPrep Kit and the DNA was extracted from the gel us ing the Sephaglass BandPrep K it (Amersham Pharmacia Biotech). The pT7.7A plasmid was from USB; BL21(DE3)pLysS E. coli cells were from Novagen Inc. Assay for GO activity GO activity was assayed using a Hansatech oxygen electrode equipped with a thermostat to measure the oxygen consumption at p H 8.5 and 25 °Cwith10m M sarcosine as substrate. One unit of GO is defined as the amount of enzyme that converts 1 lmol of substrate (sarcosine or oxygen) per minute at 25 °C. The pH effect on GO activity and s tability was dete rmined using a multicomponent buffer: 15 m M Tris, 15 m M sodium carbonate, 1 5 m M phosphoric acid, 250 m M potassium chloride and 1% (v/v) glycerol. The high [KCl] was used to buffer against minor changes i n ionic strength at different pH values. These buffers were adjusted to the appropriate pH with HCl or KOH. To assess the pH stability, activity was determined 30 min after incubation at the different pH values. For thermal inactivation, GO samples in 50 m M potassium phosphate buffer, pH 7.0, containing 10% (v/v) glycerol were incubated in a temperat ure range of 4–6 0 °C. Aliquots were withdrawn after 30 min of incubation and a ssayed for GO activity. Protein concentration was measured using the Bradford protein assay. Cloning of B. subtilis GO cDNA and DNA manipulation The cloning an d transformation techniques u sed were essentially those described by Sambrook et al.[5].Genomic DNA from B. subtilis cells strain B168 was a generous gift of A. Galizzi (Universita ` di Pavia, Italy ). PCR amplification of the yjbR gene encoding GO was obtained using Vent R DNA polymerase ( New England BioLabs) and t he fol- lowing oligonucleotides: YJBr-up (5¢-GCCAT GAATTC GCGCTATGAAAAGGCATTATGAAGCAGTGG-3¢) derived from the 5¢ end and YJBr-down (5¢-CCGAT GAATTCCATCATATCTGAACCGCCTCCTTGCG-3¢) derived from the 3¢ end of nucleotide sequ ence of yjbR gene (the sequence recognized by EcoRI restriction enzyme is underlined). This amplification yielded a product of 1139 bp representing the entire GO gene. The PCR p roduct was digested with t he restriction enzyme EcoRI, i solated by agarose gel electrophoresis and inserted i n the unique EcoRI site of the multiple cloning site of the expression vector pT7.7 downstream of t he T7 RNA polymeras e promoter t o produce the recombinant p lasmid pT7-GO. The correct orientation of the insert was checked by restriction digestion with the e nzyme HindIII. Both strands of the resulting plasmid were automatically sequenced: the nucleotide sequence was identical to t he known sequence of the yjbR gene [1]. Expression and purification of GO in E. coli The pT7-GO expression plasmid was amplified in t he E. co li strain JM109 and then transferred, for p rotein production, to the host BL21(DE3)pLysS E. coli strain. Cells carrying the recombinant plasmid were grown at 37 °CinLuria– Bertani, 2 · Luria–Bertani, 2 · YT [5] or terrific broth media containing ampicillin (100 lgÆmL )1 final concentra- tion) and c hloramphenicol (34 lgÆmL )1 final concentra- tion), and induced at selected D 600 values by adding isopropyl thio-b- D -galactoside. After induction, the cells were grown at 30 °C and collected at various times (from 1 to 24 h) by centrifugation. Crude extracts were prepared by French Press lysis (three cycles at 1000 p.s.i.) of cell suspensions obtained by r e-suspending the frozen cell paste with 50 m M sodium pyrophosphate buffer, pH 8.5, con- taining 5 m M EDTA, 0.2 l M FAD, 5 m M 2-mercaptoeth- anol, 0.7 lgÆmL )1 pepstatin, 1 m M phenylmethanesulfonyl fluoride and 10 lgÆmL )1 DNase (in a ratio of 2–3 mL of lysis buffer per gram of E. coli cells). The i nsoluble fraction of the l ysate w as removed by centrifugation a t 39 000 g for 40 min at 4 °C. The cell homogenate, obtained by French P ress lysis of  34 g of E. coli cells under the conditions reported a bove, was precipitated with ammonium sulfate at 30% of saturation (164 gÆL )1 ). The supernantant was then brought to 45% of saturation (86 gÆL )1 ). After centrifugation, the protein pellet was re-suspended a nd dialysed against 5 0 m M potassium phosphate buffer, pH 7.0, 2 m M EDTA, 1 0% glycerol a nd 5 m M 2-mercaptoethanol (buffer A). After dialysis and c entrifugation, the enzyme solution w as ap plied to a DEAE-Sepharose Fast-Flow c olumn (1.6 · 21 cm) and eluted with a 10–20% linear gradient using buffer A to which 1 M NaCl was added. Fractions containing GO activity were pooled and concentrated using a n Amicon cell concentrator equipped with a YM30 membrane. The sample was then loaded onto a hydroxyapatite column (1.6 · 12 cm) and GO eluted, using a 50 m M potassium phosphate buffer, pH 6.5, 2 m M EDTA and 10% glycerol. The f ractions containing GO activity were pooled and the pH was adjusted with sodium carbonate to pH 7.5. After the addition of 0.5 M sodium chloride (final concentration) the sample was applied to a phenyl-Sepharose CL-4B column (1.6 · 22 cm) equilibrated in 50 m M potassium phosphate buffer, pH 7.5, 0.5 M sodium chloride, 2 m M EDTA, and 5 m M 2-mercaptoethanol. The boun d enzyme was s ubsequently eluted with 50 m M potassium phosphate, pH 7.5, 5 m M 2-mercaptoethanol and 2 m M EDTA. The fractions containing GO activity were concentrated and stored at )20 °C. HisGO preparation, expression and purification The G O DNA obtained by EcoRI digestion of the original pT7-GO plasmid (see above) was inserted into the EcoRI site of the pT7-DBam/Hind plasmid [6]. The resulting recombinant plasmid, defined as pT7-HisGO, encodes for an additional N -terminal sequence c ontaining one methi- onine and six histidine residues (Fig. 1). The correct insertion of the GO gene was checked by digestion with the restriction enzymes NdeIandBamHI/ScaI and by a PCR reaction, using the XbaI [6] and YJBr-down oligonu- cleotidesasaprimer. The pT7-HisGO expression plasmid was transferred to the host BL21(DE3)pLysS E. coli strain for protein production. Recombinan t cells were grown at 3 7 °Cin 2 · Luria–Bertani medium containing ampicillin and Ó FEBS 2002 A His-tagged chimeric glycine oxidase (Eur. J. Biochem. 269) 1457 chloramphenicol (100 lgÆmL )1 and 34 lgÆmL )1 final con- centration, respectively) and induced at D 600 ¼ 0.8 by adding isopropyl thio-b- D -galactoside at a final concentra- tion of 1 m M . The cells were then grown at 30 °Cand collected after 24 h by centrifugation. Crude extracts (prepared as described above) were added of 1 M NaCl and 1 m M imidazole (all final concentrations) to improve the interaction specificity in the affinity chromatography step. The enzyme solution w as then applied to a HiTrap chelating affinity column (5 mL) equilibrated with 50 m M sodium pyrophosphate buffer, pH 7.2, containing 1 M NaCl, 20 m M imidazole and 5% glycerol, using an A ¨ KTA-FPLC system. The bound enzyme was eluted with 50 m M sodium pyrophosphate buffer, pH 7.2, containing 500 m M imidazole and 5% glycerol (elution buffer). The fractions c ontaining GO activity were pooled and l oaded o n a PD10 column equilibrated with 5 0 m M sodium pyro- phosphate b uffer, pH 8.5, containing 2 m M EDTA, 5 m M 2-mercaptoethanol and 10% glycerol. Substrate specificity Two d ifferent methods were u sed to i nvestigate the substr ate specificity of GO. Activity stain using iodonitrotetrazolium chloride (INT) on protein samples separated by native PAGE. After the electrophoretic separation, each single lane was incubated for2hinthedarkat37°C in a solutio n containing 75 m M sodium pyrophosphate, pH 8.5, 100 m M substrate, 5 l M FAD a nd 0.1 mgÆmL )1 INT (dissolved in pure ethanol). The activity was revealed as a pink band on the gel in the position corresponding to the G O. Spectrophotometric determination of GO activity meas- uring the hydrogen peroxide produced in the presence of different substrates using a 96-well ELISA plate. Each assay well has 200 lL of a solution containing 10 m M or 90 m M substrate, 0.32 mgÆmL )1 o-dianisidine and 10 UÆmL )1 of horseradish peroxidase in 90 m M sodium pyrophosphate, pH 8.5. The increase in absorbance at 440 nm was followed using a Mete rtech 960 spectropho- tometer (extinction coefficient of o-dianisidine product was 13 m M )1 Æcm )1 ). Western blot analysis, N-terminal sequencing and sequence comparison Western blot analysis was performed on total E. coli and crude protein extracts. Proteins were separated on 12% SDS/PAGE [7] and transferred electrophoretically to nitrocellulose membranes (Millipore) [8]. GO was detected by immunostaining using rabbit anti-GO Ig and visual- ized using anti-(rabbit IgG) Ig alkaline phosphatase conjugate with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue-tetrazolium chloride. The anti-GO Ig were raised in rabbit with 0 .5 mg of pure GO a s antigen according to a standard protocol (Davids Biotechnologie, Germany). The N-terminal sequence of the recombinant GO was determined on a purified soluble protein sample using an automated protein sequencer (Procise Model 492, Applied Biosystems). The BLAST program (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi) [9] was used to search for proteins showing sequence similarity. Multiple sequence alignments were performed with the CLUSTALW program (http://npsa-pbil.ibcp.fr/cgi-bin/align_clustalw.pl) [10]. RESULTS Cloning of B. subtilis GO DNA and protein sequence comparison To clone the gene encoding the B. subtilis GO, the genomic DNA was amplified by PCR using the oligonucleotides derived from the sequence of the yjbR gene of GO from B. subtilis [1]. Currently, t here are three proteins in the nonredundant Protein Data B ank that have been classified asGO.InadditiontotheenzymefromB. subtilis,the proteins from Bacillus halodurans and Thermoplas ma volcanium (accession nos BAB05153 and NP_111169, respectively) have been also included in the database. The overall sequence identity w ith B. subtilis GO is modest (27% an d 22%, respectively), although a higher conserva- tion is evident at their N-termini (containing the Rossman fold fingerprint m otif GXGXXG, w hich is in volved in binding of the ADP moiety of FAD) [11] and for the 70 residues at their C-termini (for th is latter region a 31–40% of identity and a 53–61% of sequence similarity has been calculated). The r esults of comparison of the amino-acid sequence of B. subtilis GO with the sequences available in the Data Bank are reported in Table 1. The greatest similarity was found between GO and the b subunit of heterotetrameric sarcosine oxidase (TSOX), sarcosine dehydrogenase ( SDH) and dimethylglycine dehydrogenase (DMGDH). In all cases, the N-terminal region corresponds to the flavin- binding domain a nd shows the highest sequence conserva- tion (3 2–48% of identity when only the starting 50 residues Fig. 1 . Physical map of the Bacillus subtilis GO expression plasmid pT7-HisGO . The1.13kbofGOcDNAwasinsertedintotheEco RI site of the MCS of pT7- DBam/Hind plasmid [6]. The original ATG starting codon is shown in b old and the ATG c odon corresponding to the Met1 of t he wi ld- type G O in it al ic. 1458 V. Job et al. (Eur. J. Biochem. 269) Ó FEBS 2002 are considered). A considerable homology was also observed with the sequences of monomeric sarcosine oxidases (MSOX), pipecolate oxidase (PIPOX), DAAO and D -aspartate oxidase (DASPO). P IPOX is a m ammalian enzyme that plays a significant role in brain metabolism, where L -pipecolate acts as a neuromodulator. DAAO has been proposed to regulate the levels of D -serine in b rain, a D -amino acid that modulates NMDA neurotransmission. A partial sequence comparison between GO and SOX or DAAO proteins [12,13] of t he regions containing the h ighly conserved residues that play a key role in catalysis is reportedinFig.2A,B. Table 1. BLAST results o f s earch for ide ntity an d homology with B. subtilis GO. The p ercentages refer t o t he 369 amino acids of GO. Th e h omology score is considered the sum of identical and s trongly similar a mino acids. Th e proteins i dentifi ed by the code ÔNP_Õ have been recognized following the complete sequence of the genome of the correspon ding organism; therefore, their activity was never proven. TSOX, b subunit of hetero- tetrameric sarcosine oxidase. Entry code Organism Protein Identity (%) Homology (%) BAB05153 Bacillus halodurans GO 27 51.8 NP_111169 Thermoplasma volcanium GO 21.7 48.0 NP_107418 Mesorhizobium loti TSOX 24.4 45.5 NP_126006 Pyrococcus abyssi TSOX 26.8 49.9 P40875 Corynebacterium sp. P-1 TSOX 23.8 48.8 P23342 Bacillus sp. NS-129 MSOX 21 43 P40859 Bacillus sp. B-0618 MSOX 20.6 43.4 P24552 Fusarium solani DAAO 18.4 39.3 P80324 Rhodotorula gracilis DAAO 18.4 38 P31228 Bos taurus DASPO 18.4 39 Q63342 Mus musculus DMGDH 24.7 49 NP_106044 Mesorhizobium loti SDH 27.6 49.6 NP_057602 Homo sapiens PIPOX 21.4 46.9 Fig. 2. Details of the multiple sequence alignment f or GO from B. subtilis with sequences of S OX (A) and DAAO (B) and comparison of the 3D structure o f t he active site with those o f other enzymes (C). Residues marked with (*) i ndicate identity ( :) indicate strongly similar, and ( .)indicate weakly si milar. The b oxes identify the amino acids present in the active s ite of M SOX [12] and of DAAO [15,16]. (C) C omparison of t he active site ofthe3D-structuremodelofGOwiththeactivesiteofMSOXfromBacillus subtilis (right) [12] a nd D A AO f rom Rh odotorula gracilis (left) [15]. Ó FEBS 2002 A His-tagged chimeric glycine oxidase (Eur. J. Biochem. 269) 1459 Using the Swiss-Model software at the EXPASY website [14], a model of the 3D structure of G O has been predicted using its protein sequence and the 3D coordinates of Rhodotorula g racilis DAAO as template [15]. D ue to the limited sequence homology between these t wo enzymes, the GO model only extends from Y240 to G313. A comparison of the active s ites of MSOX and D AAO with the active site of the model derived for GO ( Fig. 2C) confirms that Y246 is highly c onserved and is located a t a position resembling that of Y223 in DAAO and Y254 in MSOX. Even the arginine residue (R285 in yeast D AAO) which e lectrostatically interacts with t he a-carboxylate of the D -amino acid in DAAO [15,16] is conserved in GO (R302, see Fig. 2C). On the other hand, and according to the sequence comparison, M261 in GO appears t o be located at the position occupied by Y238 and H 269 in DA AO and MSOX, respectively. This second active site residue, which is involved in the recog- nition/binding of the substrate in both MSOX a nd DAAO [12,15], is not conserved in GO and probably represents a key element for defining its substrate specificity. The limited (sequence) similarity/homology between GO, DAAO, MSOX and TSOX is also confirmed by t he results obtained in Western blot experiments using monospecific anti-DAAO [17] and a nti-GO Ig. The antibodies raised against pure B. subtilis GO did not recognize DAAO a nd MSOX (a faint band is only observed when a large amount of TSOX is used, data not shown). Analogously, anti- DAAO IgG did not recognize GO, MSOX or TSOX. Expression of B. subtilis GO gene in E. coli and GO purification The DNA encoding for G O was inserted in the EcoRI site of the multiple cloning site of the expression vector pT7.7 downstream of the T7 RNA polymerase promoter to produce the recombinant plasmid pT7-GO. Because of the presence of an ATG c odon upstream of the cloning site, t his procedure produces a fusion protein; six additional residues are a dded a t t he N-terminus of the protein before the original starting methionin e. The new ATG starting codon is positioned 8 bp downstream from the ribosomal binding site. The recombinant plasmid pT7-GO was used to transform BL2 1(DE3)pLysS E. coli cells. A significant increase in GO synthesis was observed immediately after the addition of 1 m M isopropyl thio-b- D -galactoside in cell cultures transformed with the pT7-GO expression vector and grown on Luria–Bertani m edium, as indicated by native PAGE and GO activity staining. The highest level of GO expression and GO specific activity was obtained 24 h after induction in the mid-log growth phase of a c ulture grown on Luria–Bertani medium. No activity was detected in E. coli extracts from cells carrying the vector without the 1.13-kb GO DNA (data not shown). The GO expression depends on the isopropyl thio-b- D -galactoside concentration: West- ern blot experiments show that the maximal amount of immunoreactive GO in soluble crude extract, as well as in the total cell, was achieved at a n isopropyl thio-b- D - galactoside concentration o f 0.5 m M . Analogously, the higher GO fermentation yield (in terms of GO units per gram of cell p aste and GO s pecific activity) was obtained at the same concentration of inducer (data not shown). A comparison of the amount of immunoreactive GO in the total cell with the amount of GO in the crude extract on Western b lot indicates t hat GO is almost t otally expressed i n a soluble form. Under the best conditions, the GO expression reached  1UÆg )1 cell and 4 U ÆL )1 of fermen- tation broth (with a G O specific activity in the crude extract of 0.006 UÆmg )1 protein). GO accumulated to 1% of total E. coli soluble proteins in the crude extract. Recombinant GO was purified from E. coli cells by precipitation with ammonium sulfate twice and then a three-step chromatography procedure (see Materials and methods). Ab out 13 mg of enzyme (90% homogenous) were obtained starting from 3 4 g of cell paste (Table 2). The total yield of the purification was about 20%. The N-terminal sequence of the first eight amino acid s of the recombinant GO was determined. This sequence is unique and was identical to that inferred from the nucleotide sequence [1]; protein sequencing o f 0.2 nmol of the purified enzyme resulte d in the A RIRAMKR s equence, confirming the presence of five of the six additional residues at the N-terminus of recombinant chimeric GO (the starting methionine is not present). The recombinant GO produced in E. coli was purified as a holoenzyme with spectral p roperties typical of the flavin-containing oxidases (absorbance maxima at 457 nm and 376 nm and a A 274 /A 456 ¼ 9). Na tive GO is a 180-kDa homotetramer, as determined by SDS/PAGE (46.6 ± 1.3 kDa) and gel permeation chromatography on a Superdex 200 column (187.6 ± 2.3 kDa). The recombin- ant enzyme catalysed the oxidation of sarcosine and glycine (specific activity of 0.57 and 0 .43 UÆmg )1 protein, at 25 °C). Table 2. Purifica tion of recombinant B. subtilis glycine oxidase from BL21(DE3)pLysS E. coli cells c ontai ning the pT7-GO (top) a nd pT7-HisGO (bottom) plasmid. Starting material was 34 g of E. coli c ell paste obtaine d from a 10-L fe rmentation (GO ) and 38 g of E. coli cell paste obtained from a 6-l fermentation (HisGO). Step GO activity (U) Protein content (mg) Specific activity (UÆmg protein )1 ) Purification index Yield (%) GO Crude extract 38.2 6617 0.006 1.0 100 (NH 4 ) 2 SO 4 precipitation 25.0 2984 0.008 1.4 65.4 DEAE-Sepharose 23.4 512 0.046 7.9 61.3 Hydroxyapatite 14.6 112 0.130 22.4 38.1 Phenyl-Sepharose 7.5 13.1 0.568 98.0 19.5 HisGO Crude extract 86.6 2188 0.039 1.0 100 HiTrap Chelating 86.3 80 1.06 27.2 98 1460 V. Job et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Addition of exogenous FAD or FMN to the assay mixture did not increase enzyme activity, indicating a tight binding of the coenzyme to t he protein moiety. Preparation, overexpression and purification of the chimeric His-tagged GO The scheme of the strategy devised for obtaining a plasmid coding for the chimeric His-tagged GO is reported in the Materials and methods section; the map of the resulting expression vector pT7-HisGO is given in Fig. 1. In these experiments the conditions for the expression of GO in E. coli BL21(DE3)pLysS cells were optimized. Cells were grown at 37 °C and induced with 1 m M isopropyl thio-b- D - galactoside at an D 600 ¼ 0.8; the growth temperature was then decreased to 30 °C a nd the cells were harvested 24 h after induction. The protein was overexpressed under the reported co nditions and was totally soluble and thus recovered in the crude extract. In addition, the chimeric HisGO was entirely present as an active holoenzyme. Therefore, the addition o f exogenous FAD o r FMN to the assay m ixture did n ot increase the activity a t all. The enzyme expression was about 1 UÆg )1 of wet weight ( 2.1 mg GO/gofwetweightcelland 4% of t otal E. coli soluble proteins in the crude extract) and the fermentation yield w as  14.4 UÆL )1 per culture. The c rude extract from induced E. coli cells was loaded onto a HiTrap chelating affinity column following the addition of 1 M sodium chloride and 1 m M imidazole (all final concentrations). Under the elution conditions we employed (see Materials and methods), the engineered G O was selectively e luted as a single resolved peak (data not shown). The final preparation was 95% homogeneous. The sequence analysis (MHHHHHHMARIRA) of the purified enzyme confirmed the presence of the basic additional sequence (the starting methionine plus the six histidine tag plus the additional s ix residues of the chimeric protein) at the N-terminus, which clearly does not impair either the enzymatic activity or the coenzyme binding. The purification of the His-tagged chimeric GO and, for comparison, that of the wild-type enzyme is reported in Table 2 . From this table it is apparent that the purification procedure of t he engineered GO is far less time-consuming (the final G O preparation is obtained in few hours s tarting from the E. coli cell paste). Rema rkably, a nd due to the fast procedure used, we obtained a f ully active holoenzyme with a specific activity that was twofold higher than that of the wild-type recombinant enzyme (1.06 vs. 0.57 UÆmg )1 pro- tein). In addition, the total recovered enzyme a ctivity obtained starting from a similar amount of E. coli cell paste are higher for the HisGO than for the wild-type enzyme. Properties of the His-tagged chimeric GO The molecular mass of HisGO, as determined by SDS/ PAGE, is slightly higher than the value calculated from the amino-acid sequence (49.4 ± 1.1 k Da vs. 42.66 kDa, respectively). U nder native conditions, the molecular mass of the His-tagged GO is 166.4 ± 11.1 kDa, as determined by gel permeation chromatography. The elution volume (R t ¼ 13.4 ± 0.2 mL) is not dependent on the protein concentration in the range 0.01–12 mgÆmL )1 . T he result is consistent with the presence in solution of a stable homotetramer. The theoretical isoelectric points of GO and HisGO are 6.14 and 6.34, respectively. However, we were not able to determine this under any of the present experimental conditions due to the instability of the protein at a pH < 7 (see below). The purified holoenzyme shows the typical absorbance spectrum of F AD-containing proteins, with two well-resolved peaks in the visible region (at 458 nm and 378 nm, see Fig. 3). Anaerobic addition of an excess of glycine resulted instantaneously in the forma- tion of reduced enzyme flavin, as shown by its spectrum with a maximum at 346 nm (Fig. 3). The spectral p roperties of oxidized and reduced recombinant GO are significantly different from those previously reported, e.g. the reported flavin reduction following addition of 10 m M sarcosine resulted in a 20% decrease of the  455 nm peak [2], which is not compatible with full flavin reduction. Plotting GO activity (on sarcosine as substrate and using both GO a nd HisGO ) over t he t emperature ra nge 15–60 °C shows an increase in a ctivity with no evidence f or any plateau or decrease up to 60 °C (Fig. 4A). HisGO shows good temperature stability in t he range 4–35 °C when the samples are incubated for 30 min at pH 7 .0. When the temperature is increased to above 35 °C, however, a sharp decrease in enzyme stability is evident (Fig. 4A): a T m of 46 °C was seen. T he activity is maximal a t a bout pH 10, but falls off rapidly above pH 10, probably due to instability (Fig. 4B). The stability is maximal in the 7–8.5 pH range. Below pH 6.5 and above pH 9.5, a considerable decrease in stability was observed (Fig. 4B). These profiles were iden- tical when GO was used instead of HisGO (data not shown). Both GO and HisGO enzymes are stable for months when stored at )80 °CandatapH ¼ 7.5. Substrate specificity Purified GO shows a similar specific activity on sarcosine and glycine as substrates (1.06 and 1.00 UÆmg )1 protein, respectively). Because of the sequence similarity of GO with Fig. 3. Absorbance spectrum of the purified HisGO ( 12 l M )inthe oxidized form (1) and in the reduced form (2), as obtained under anaerobic conditions by t he addition of 20 m M glycine in 5 0 m M potas- sium phosphate buffer pH 7.0, 5 m M 2-mercaptoethanol, 2 m M EDTA and 10% glycerol. Ó FEBS 2002 A His-tagged chimeric glycine oxidase (Eur. J. Biochem. 269) 1461 a number of flavoenzymes (see Table 1), the substrate specificity of the reaction catalysed by GO has been investigated using several compounds that are known to be substrates of DAAO, DASPO, SOX, DMGDH and PIPOX. Rapid s creening was performed by measuring the increase in absorbance at 440 nm following the horseradish peroxidase assay coupled with o-dianisidine using a 96-well ELISA plate. The results, reported as a percentage with respect to t he absorbance change observed with sarcosine as 100%, a re shown in Fig. 5. From t his it is evident that GO possesses a wide substrate specificity. In addition to sarcosine and glycine, even N-e thylglycine, ethylglycine ester, D -alanine, D -2-aminobutyrate, D -proline, D -pipecolate and N-methyl- D -alanine are good substrates; here, a lower activity is observed on branched and polar D -amino acids (e.g. D -Val, D -Ile, D -Leu, D -His, and D -Arg). GO is strictly stereospecific as it only catalyses the oxidation of the D -isomer, as further demonstrated by the INT staining assayreportedinFig.5,inset. A comparison of t he activity measured u sing a similar amount (5 mU) of GO, DAAO and MSOX on various substrates showed that GO and DAAO oxidize D -proline, D -alanine and D -2-aminobutyrate with similar relative efficiencies. Analogously, GO and MSOX show a fairly similar activity o n s arcosine and N-ethylglycine. In contrast, an appreciable activity on glycine, glycine-ester a nd D -pipecolic acid was only observed using GO. Interestingly, D -proline i s t he only amino acid in which the a-amino group is involved in a covalent bond with the side chain and it is the only compound that was oxidized by all three enzymes used. I t i s also the only D -amino ac id that is oxidized efficiently by both D AAO and D -aspartate oxidase [18]. CONCLUSIONS The present data demonstrate the successful overexpression of a chimeric B. subtilis GO in E. coli (upto3.9%oftotal soluble protein) using two different expression systems. In particular, we produced a recombinant GO c ontaining a polyhistidine tag at the N-terminus, that can be effi ciently purified in a single step by metal-chelate chromatography, producing a stable holoenzyme with a high specific activity and a 98% yield quickly and simply. Its properties (aggregation state, absorbance spectrum and stability) are identical to those of native GO, but the specific activity is twofold higher. The spectral properties of GO in the oxidized and r educed form (Fig. 3) unambiguously identify it as a flavoenzyme. A comparison of the expression level and purification yield obtained with the previo us procedure [2] is not feasible because no quantitative data have been previously reported. Sequence comparison and Western blot analyses show that GO is not highly similar to any known flavo- enzyme, although the observed sequence homology a nd Fig. 5. Assay of G O activity on a number of compounds using the horseradish peroxidase assay and o-dianisidine. The values are reported as a percentage of the absorbance change a t 440 nm obtained with sarcosine (referred a s 100%) after 3 h of incubation a t pH 8.5, pH 5.8 (a) or pH 9.7 (b). Inset: assay o f GO activity on various compounds using t he activity stain w ith INT on enzyme sampl es (6 lg) separated by native PAGE in t he presence of: (1) sa rcosine; (2) glycine; (3) N-ethylglycine; (4) N,N-dimethylglycine; (5) D -alanine; (6) L -alanine; (7) D -proline; (8) L -proline; (9) D -valine; (10) L -valine, as substrate. Fig. 4. Temperature–activity and temperature stability profiles (A) and pH–activity and pH stability profiles of HisGO (B). Enzyme activity (d) was assayed in the tem perature range 15 –60 °C. For thermostability (s) e nzym e samples were incubated at th e indicated t empe rature for 30 minin50 m M potassium phosphate, pH 7.0, 10% glycerol a nd then assayed using the O 2 consumption a ssay at 25 °C . Data are expressed as per cent o f enzyme activity in t he standard assay; the l ines through the data points have been obtained by smooth fitting. SEM is the average of three determinations. (B) pH–activity and pH stability profiles of HisGO. Enzyme a ctivity (d) was assayed in the pH range 4.8–12. For thermostability ( s) enzyme samples we re incubated a t the indicated pH for 30 min in the multicomponent buffer (see Materials and methods) and the n assayed usin g the stand ard O 2 consumption assay at 25 °C. Data are exp ressed as per cent of enzyme activity in the standard assay; th e lines through t he data points have be en obtained by smooth fitting. The SEM is the average of three determinations. 1462 V. Job et al. (Eur. J. Biochem. 269) Ó FEBS 2002 conservation of active s ite residues suggest that GO would exhibit significant structural similarity with enzymes acting on sarcosine or sarcosine analogues (monomeric and tetra- meric sarcosine oxidases and s arcosine and dimethylglycine dehydrogenases), on D -amino acids ( DAAO and DASPO) and on p ipecolic acid (PIPOX) (see Table 1). T he metab olic role of GO in B. subtilis and its physiological substrates are unknown. Its substrate specificity partially overlaps with that of DAAO and SOX and indicates an oxidative role of this oxidase in B. subtilis on a wide r ange of compounds. The c loning and overexpression of the B. subtilis GO also provides the basis for kinetic, site-directed mutagenesis and structural stu dies and, thu s, identification of the underlying structure of the catalytic properties and substrate specificity of this novel flavoenzyme. This may, in turn, make it possible to engineer the GO activity for use as a biocatalyst. 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