Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Alcohol dehydrogenases are able to catalyze the conversion of alcohols to aldehydes or ketones, simultaneously reducing the cofactor NAD+ or NADP+ to NAD(P)H. Because of the high costs of these pyridine cofactors, in situ cofactor regeneration is required for preparative applications in order to reach turnover numbers that are sufficient for economically viable processes. Here we present the development of a process for the enantioselective oxidation of rac-1-phenylethanol to acetophenone, applying an alcohol dehydrogenase coupled with an NAD(P)H oxidase for the enzymatic cofactor regeneration, which is active towards NADH as well as NADPH. The reaction system was investigated in view of various influential parameters with main focus on the external oxygen supply. We could show that a gassed stirred tank reactor is a promising reactor concept to run NAD(P)H oxidase-coupled alcohol dehydrogenase oxidations, including the possibility to scale-up the system.
An l -aminopeptidase of Pseudomonas putida , used in an industrial process for the hydrolysis of d,l -amino acid amide racemates, was purified to homogeneity. The highly l -enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and α-H amino acid amides, e.g., l -phenylglycine amide.
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
The α-amino acid ester hydrolase fromAcetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in β-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate appeared to be an active site titrant and was used to label the α-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser205, situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser205with the catalytic nucleophile of some α/β-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg85, Asp86, Tyr143, Ser156, Ser205, Tyr206, Asp338, His370, Asp509, and His610). Mutation of Ser205, Asp338, or His370 to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the α-amino acid ester hydrolase has an α/β-hydrolase fold structure with a catalytic triad of Ser205, Asp338, and His370. This distinguishes the α-amino acid ester hydrolase from the Ntn-hydrolase family of β-lactam antibiotic acylases. The α-amino acid ester hydrolase fromAcetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in β-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate appeared to be an active site titrant and was used to label the α-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser205, situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser205with the catalytic nucleophile of some α/β-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg85, Asp86, Tyr143, Ser156, Ser205, Tyr206, Asp338, His370, Asp509, and His610). Mutation of Ser205, Asp338, or His370 to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the α-amino acid ester hydrolase has an α/β-hydrolase fold structure with a catalytic triad of Ser205, Asp338, and His370. This distinguishes the α-amino acid ester hydrolase from the Ntn-hydrolase family of β-lactam antibiotic acylases. α-amino acid ester hydrolase p-nitrophenyl-p′-guanidino-benzoate p-nitrophenol d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid electrospray mass spectrometry high-pressure liquid chromatography dimethylformamide Protein Data Bank The α-amino acid ester hydrolases have been known for their applicability in the biocatalytic synthesis of semisynthetic β-lactam antibiotics since 1972 (1Takahashi T. Yamazaki Y. Kato K. Isona M. J. Am. Chem. Soc. 1972; 94: 4035-4037Crossref PubMed Scopus (60) Google Scholar). These enzymes can hydrolyze the amide bond that connects the acyl side chain to the β-lactam nucleus. Starting from esterified acyl precursors, they can also catalyze the reverse reaction. Remarkable features of these enzymes are the ability to accept charged substrates such as α-amino acid esters, the preference for esters over amides, and the low pH optimum (pH 6.2) (2Takahashi T. Yamazaki Y. Kato K. Biochem. J. 1974; 137: 497-503Crossref PubMed Scopus (45) Google Scholar, 3Blinkovsky A.M. Markaryan A.N. Enzyme Microb. Technol. 1993; 15: 965-973Crossref PubMed Scopus (35) Google Scholar). Despite these attractive properties, a gene encoding an α-amino acid ester hydrolase (AEH)1 was only recently cloned and characterized (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). Thus far, all the known β-lactam antibiotic acylases, such as penicillin G acylase (5Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C.E. Nature. 1995; 373: 264-268Crossref PubMed Scopus (425) Google Scholar), penicillin V acylase (6Suresh C.G. Pundle A.V. Siva Raman H. Rao K.N. Brannigan J.A. McVey C.E. Verma C.S. Dauter Z. Dodson E.J. Dodson G.G. Nat. Struct. Biol. 1999; 6: 414-416Crossref PubMed Scopus (106) Google Scholar), and cephalosporin acylase (7Kim Y. Yoon K.-H. Khang Y. Turley S. Hol W.G.J. Structure. 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), belong to the Ntn-hydrolase family. However, protein data base searches showed no homology of the AEH of Acetobacter turbidans with known β-lactam antibiotic acylases. The N-terminal amino acid sequence of the AEH was determined; it revealed a signal sequence, but no N-terminally located Thr, Ser, or Cys, characteristic for members of the Ntn-hydrolase family, was found. It was therefore postulated that the AEHs belong to a new class of β-lactam antibiotic acylases (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). An alignment of the AEH sequence with those of homologous proteins showed the presence of the active site serine consensus motif GXSYXG (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar), which is described for the X-prolyl dipeptidyl aminopeptidases (8Chich J.-F. Chapot-Chartier M.-P. Ribadeau-Dumas B. Gripon J.-C. FEBS Lett. 1992; 314: 139-142Crossref PubMed Scopus (29) Google Scholar). No x-ray structure of the aminopeptidases is known, but they are members of a group of proteins that belong to the prolyl oligopeptidase family. Of this family two structures have been solved, which both contain an α/β-hydrolase fold (9Fülöp V. Böcskei Z. Polgár L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 10Medrano F.J. Alonso J. Garcı́a J.L. Romero A. Bode W. Gomis-Rüth F.X. EMBO J. 1998; 17: 1-9Crossref PubMed Scopus (70) Google Scholar) and have a catalytic triad of Ser, Asp, and His. Therefore, it is possible that the X-prolyl dipeptidyl aminopeptidases and hence AEH also have a catalytic triad. This assumption is further supported by the identification of a catalytic triad in the recently solved crystal structure of a cocaine esterase (11Larsen N.A. Turner J.M. Stevens J. Rosser S.J. Basran A. Lerner R.A. Bruce N.C. Wilson I.A. Nat. Struct. Biol. 2001; 9: 17-21Crossref Scopus (105) Google Scholar) that is also related to AEH. Earlier experiments with inhibitors already suggested the importance of a histidine for the catalytic activity of AEH (12Ryu Y.W. Ryu D.D.Y. Enzyme Microb. Technol. 1988; 10: 239-245Crossref Scopus (12) Google Scholar). However, common serine hydrolase inhibitors such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, or Pefabloc SC showed no inhibition of AEH activity (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar, 12Ryu Y.W. Ryu D.D.Y. Enzyme Microb. Technol. 1988; 10: 239-245Crossref Scopus (12) Google Scholar). On the other hand, inhibition was observed with the serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate (p-NPGB), but the inhibition was incomplete, which left uncertainty about the catalytic role of a serine in AEH (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). In this study, we used active site labeling, site-directed mutagenesis, and sequence analysis to demonstrate that AEH is a member of a class of β-lactam antibiotic acylases that belongs to the α/β-hydrolase fold family and possesses a classical catalytic triad of Ser, Asp, and His. The chromogenic substrated-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) was obtained from Syncom (Groningen, The Netherlands). Phenylglycine methyl ester, 7-aminodesacetoxycephalosporanic acid, and cephalexin were provided by DSM Anti-infectives (Delft, The Netherlands). All chemicals used in DNA manipulation procedures were purchased from Roche Diagnostics GmbH and used as recommended by the manufacturer. The oligonucleotides for the cloning of the aehA gene and introduction of point mutations were synthesized by Eurosequence B.V. (Groningen, The Netherlands). Escherichia coli TOP10 (Invitrogen) was used for cloning derivatives of pBAD/Myc-HisA (Invitrogen) and pTrcHisB (Invitrogen). E. coli strain BL21(DE3)pLysS (Promega, Madison, WI) was used for cloning derivatives of pET28 (Promega). The E. coli strains were grown at 30 °C for plasmid isolation. For expression, strains with pTrcHisB and pET28 derivatives were grown on LB medium at 30 °C and directly induced with isopropyl-β-d-thiogalactopyranoside (0.4 mm). The antibiotics ampicillin and kanamycin were added to the media at 100 and 50 μg/ml, respectively. To clone aehA in theNcoI and HindIII site of pBAD/Myc-HisA, resulting in pBADAT, the NcoI restriction site was first removed from the gene cloned in pAT (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). This was accomplished by PCR using the sense primer 5′-GAACTGCCTGTGTCTATGGATATTTTCCGGGGC-3′, the compatible reverse complement primer, and the QuikChange site-directed mutagenesis kit of Stratagene (La Jolla, CA), resulting in pATdelNco. From this construct, the gene encoding AEH was amplified by PCR using two mutagenic primers to allow cloning in the NcoI and HindIII site of pBAD/Myc-HisA. The forward primer, 5′-CGCGCCACACCATGGTGGGACAGATTA-3′ (start codon shown in bold), was based on the N-terminal sequence including the signal sequence, and an NcoI site (underlined) was introduced. The reverse primer, 5′-CATACTGGCAAGCTTCTGTTTCACAACCGGGAG-3′ (theHindIII site is underlined), lacked the stop codon to allow the C-terminal attachment of the myc epitope followed by a polyhistidine region of six histidine residues (His6 tag), which are encoded on pBAD/Myc-HisA. Site-directed mutagenesis was performed on pBADAT using the QuikChange site-directed mutagenesis kit from Stratagene according to the procedure recommended by the manufacturer. When possible, a restriction site was introduced in the mutagenic primers (Table I). The PCR reaction mixture was directly used to transform chemically competent E. coli TOP10 cells. For isolation of vector, the cultures were grown overnight on LB medium at 30 °C. Mutated plasmids where checked by restriction analysis (when possible). All mutants and constructs were verified by DNA sequencing at the Department of Medical Biology of the University of Groningen (Groningen, The Netherlands).Table ISynthetic oligonucleotidesOligonucleotide sequence 5′ → 3′Restriction siteAmino acid substitutionC GAG GTT ATG GTA CCC ATG GCG GAC GGC GTG AAGRsaIR85AGTT ATG GTA CCC ATG CGG GCC GGC GTG AAG CTGRsaID86AG TTT GTA GAG GGC GGC GCT ATC CGC GTG TTT CAGY143AGC GGG AAA TAT GGC GCT CAG GGC GAT TAT GHaeIIS156AGGT ATG ACA GGG TCG GCC TAT GAG GGC TTT ACTAspIS205AG GGT ATG ACA GGG TCG TCC GCT GAG GGC TTT ACT GAspIY206AGAA CAG GGC TTG TGG GCT CAG GAA GAT ATG TGD338AG ATG GGC CCA TGG CGG GCT AGT GGG GTG AACNcoIH370ACA GAA TCC CGC CCG GCT GTG GTG ACA TAT GAA ACNdeID509AC CAT GTG TTT GCA AAA GGG GCT CGG ATT ATG GTGH610AOligonucleotides used in site-directed mutagenesis. Only the sense primers are shown. Introduced restriction sites are underlined, and sequence differences with wild type are shown in bold. Open table in a new tab Oligonucleotides used in site-directed mutagenesis. Only the sense primers are shown. Introduced restriction sites are underlined, and sequence differences with wild type are shown in bold. Wild-type and mutated AEHs were expressed in E. coli TOP10 from the pBAD/Myc-HisA-derived constructs. To obtain soluble protein, two 2.5-liter cultures supplemented with l-arabinose (0.01%, w/v) were inoculated with 1 ml of culture grown overnight at 30 °C and incubated for 64 h at 14 °C. Induced cells were harvested from the cultures by centrifugation at 5000 ×g and suspended in 50 mm sodium phosphate buffer, pH 6.2. All further steps were carried out at 4 °C. The cytoplasmic content was released by sonification, and the remaining cell debris was removed by centrifugation at 13,000 ×g for 40 min. The supernatant was added to 1 ml of nickel-agarose (Qiagen GmbH, Hilden, Germany) equilibrated with wash buffer (25 mm imidazole, 500 mm NaCl, and 50 mm sodium phosphate buffer, pH 7.4). After mixing by inversion for 90 min at 4 °C, the bed was allowed to form (20 × 8-mm bed in a polyprep chromatography column (Bio-Rad)). The unbound protein was washed from the column with 30 column volumes of wash buffer. The bound protein eluted from the column at 75–100 mm imidazole in a stepwise gradient from 50 to 200 mm imidazole, 150 mm NaCl, 50 mmsodium phosphate, pH 7.4, in 20 column volumes. The protein was brought to 50 mm sodium phosphate buffer, pH 6.2, with the use of an Econo-Pac gel filtration column (Bio-Rad). All purification steps were monitored by SDS-PAGE, and the enzymatic activity was measured with NIPGB (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). The protein concentrations were determined using the Bradford method with bovine serum albumin as the standard. Far-ultraviolet CD spectra from 250 to 190 nm were recorded on an AVIV circular dichroism spectrometer model 62A DS (AVIV Associates, Lakewood, NJ) at 25 °C using a quartz cuvette with a path length of 0.1 cm. The concentration of wild-type and mutant enzymes was 0.2 mg/ml in 50 mm sodium phosphate buffer, pH 6.2. Three separate spectra were collected per sample and averaged using a step interval of 0.5 nm/min and an averaging time of 5 s. The phosphate buffer was used as a blank and subtracted from each recording. The data were converted to mean residue ellipticity (θMRE, deg·cm2·dmol−2). From the CD spectra, the percentage of secondary structure elements was calculated using CD spectra deconvolution (CDNN Version 2.1, available on the World Wide Web). These values were standardized to 100% total structure elements. The hydrolysis and synthesis of cephalexin at 30 °C were followed by high-pressure liquid chromatography (HPLC) as described previously (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). The hydrolysis of p-NPGB was measured at concentrations varying from 0.1 to 1 mm with 1.5 μm enzyme. The release of p-nitrophenol (p-NP) was measured at 405 nm and 30 °C using a spectrophotometer (Lambda Bio 10 and software package UV WinLab; PerkinElmer Life Sciences). A stock solution of p-NPGB (10 mm) was made in dimethylformamide (DMF) and acetonitrile in a 1:4 volume ratio. The steady-state reactions were done in 50 mm sodium phosphate buffer, pH 7.0. The molar extinction coefficient of p-NP at pH 7 was determined as 9200m−1 cm−1. The pre-steady-state kinetics of p-NPGB conversion was determined using an Applied Photophysics SX17MV stopped-flow instrument. A stock solution of p-NPGB (100 mm) was made in DMF. The final concentration of DMF in the reaction mixture was ≤1%. All pre-steady-state reactions were performed in 50 mm 4-morpholinepropanesulfonic acid buffer at pH 7, with 1 mm p-NPGB. The enzyme concentration used was 1.32 or 0.66 μm (α2; 144 kDa). Progress curves (absorbance, P) were fit to Eq. 1 to obtain the amplitude (B), the first order rate constant (k obs) for the burst phase, and the velocity of the steady-state reaction (A), using the program Scientist. [P]=A×t+B(1−e−kobs·t)Equation 1 The enzyme (2.4 μm, 144 kDa) was inactivated by incubation withp-NPGB (1 mm; 1% DMF) for 15 min at 30 °C. Control experiments involved incubation under the same conditions of enzyme only and enzyme with 1% DMF. To study reactivation, the inactivated enzyme was diluted 76-fold in 15 mm NIPGB dissolved in 50 mm sodium phosphate buffer, pH 6.2. The time course of reactivation was monitored by following the hydrolysis of NIPGB at 30 °C and 405 nm. The enzyme (15.4 μm) was incubated with 0.5 mm p-NPGB in 50 mm sodium phosphate buffer, pH 6.2, 0.5% dimethylformamide, for 15 min at 30 °C. The excess ofp-NPGB was removed by dialysis against 70% formic acid. To reduce any disulfide bonds, the enzyme solution was dialyzed against 70% formic acid with β-mercaptoethanol (2 mm). After removing the β-mercaptoethanol by dialysis against 70% formic acid, the labeled protein was treated with a 100-fold molar excess of CNBr over the Met content. The reaction was allowed to proceed for 24 h at room temperature under N2 in the dark and was stopped by the addition of 10 volumes of water. The reaction mixture was freeze-dried and dissolved in HPLC eluents. The generated peptides were separated by reversed-phase HPLC using a Nucleosil-5 C18 column (4.6 × 300 mm; Alltech Associates, Inc.) at 1 ml/min in a linear gradient of 0 to 67% acetonitrile in 0.1% trifluoroacetic acid. The peptide profile was monitored at 280 nm. The control experiment involved the same conditions as described above, except that no p-NPGB was added. The peaks that were different from the control experiment were collected and rechromatographed on the same column in a linear gradient from 0 to 67% acetonitrile in 0.1% ammonium acetate, pH 5.0. The individual peaks were collected, concentrated, and injected directly into the mass spectrometer. Electrospray (ES) mass spectrometry (MS) was performed on an API3000 mass spectrometer (Applied Biosystems/MDS-SCIEX, Toronto, Canada), a triple quadrupole mass spectrometer supplied with an atmospheric pressure ionization source, and an ionspray interface (13Bruins A.P. Mass Spectrom. Rev. 1991; 10: 53-77Crossref Scopus (272) Google Scholar). The spectra were scanned in the range between m/z 400 and 1600. Tandem mass spectrometry product ion spectra were recorded on the same instrument by selectively introducing the m/z 1229.5 (singly charged unlabeled peptide) and m/z 695.9 (doubly charged labeled peptide) precursor ions from the first quadrupole into the collision cell (second quadrupole). The collision gas was nitrogen with 30 eV collision energy. The product ions resulting from the collision were scanned over a range of m/z 10 to 1395 with a step size of 0.1 atomic mass unit and a dwell time of 2 ms. PSI-Blast (14Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61065) Google Scholar) and a homology-based fold prediction program (15Huynen M. Doerks T. Eisenhaber F. Orengo C. Sunyaev S. Yuan Y.P. Bork P. J. Mol. Biol. 1998; 280: 323-326Crossref PubMed Scopus (92) Google Scholar) were used to predict the catalytic residues and the fold of AEH. The secondary structure elements of AEH were predicted using the consensus of the following programs: PSIPred (17Jones D.T. J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4562) Google Scholar), Jpred (18Cuff J.A. Barton G.J. Proteins. 1999; 34: 508-519Crossref PubMed Scopus (551) Google Scholar), and SAM-T99sec (19Karplus K. Barrett C. Hughey R. Bioinformatics. 1998; 14: 846-856Crossref PubMed Scopus (923) Google Scholar). To achieve a higher expression level and an easier purification of AEH than that obtained with a previous construct (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar) the aehA gene was cloned in pBAD/Myc-HisA (pBADAT), coupling both the myc epitope and the His6 tag C-terminally to the protein. The use of the arabinose promoter in the pBADAT plasmid resulted in an overproduction of 5-fold (1% of the total protein in cell-free extract) compared with the expression in the wild-type A. turbidans strain (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). Furthermore, with the resulting construct, the number of necessary purification steps was reduced from four to two by use of a nickel-agarose column (Table II). Two mg of >90% pure protein could be obtained from a 5-liter culture and was stable at 4 °C for at least 60 days. The attachment of the tag resulted in a 2-kDa increase in the molecular mass of each subunit of the homodimeric AEH, as is clearly visible on an SDS-PAGE gel (Fig.1). To check whether the properties of AEH had changed upon the addition of the myc epitope and the His6 tag, the kinetic parameters of the purified enzyme for cephalexin hydrolysis were measured (TableIII) and compared with those of untagged recombinant protein (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar). The K m values of both proteins appeared to be similar (0.45 and 0.34 mm, respectively). The k cat of the fusion protein is somewhat lower than that for the untagged recombinant protein (347 s−1), but the values are in the same order of magnitude. This indicates that proper folding of the recombinant protein occurs and shows that there is no dramatic influence of the additional C-terminal amino acids.Table IIPurification of N-terminal His6-tagged AEH from E. coliPurification stepTotal volumeTotal proteinTotal activity2-aCephalexin synthesis; cexU, units of cephalexin.Specific activity2-aCephalexin synthesis; cexU, units of cephalexin.PurificationRecoverymlmgcexUcexU/mgfold%Cell-free extract72312555017.61100Ni2+-agarose4.82.7359013307665Gel filtration8.023340167095602-a Cephalexin synthesis; cexU, units of cephalexin. Open table in a new tab Table IIIKinetic parameters of cephalexin hydrolysis for mutants of AEHEnzymeMean ± SE ofK mk catk cat/K mmms−1s−1/mmAEH-His60.45 ± 0.06274 ± 7609S205A<0.13-aNo conversion was observed at 1, 10, or 25 mm cephalexin; detection limit is given.Y206A4.0 ± 0.2120 ± 230D338A0.3 ± 0.10.20 ± 0.040.6H370A0.4 ± 0.10.20 ± 0.020.4R85A0.18 ± 0.06184 ± 61022S156A0.25 ± 0.08132 ± 3528H610A3-bPartially purified, approximately 30% pure.0.9 ± 0.2>69 ± 2>773-a No conversion was observed at 1, 10, or 25 mm cephalexin; detection limit is given.3-b Partially purified, approximately 30% pure. Open table in a new tab To check whether the previously observed inhibition by p-NPGB (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (30) Google Scholar) was irreversible, the enzyme was preincubated with p-NPGB and then mixed with substrate solution. Upon dilution into the NIPGB solution, the inactivated enzyme gradually reverted to the active form (Fig.2 A). After 20 min, the enzyme recovered a major part of its activity, indicating that the inactivation by p-NPGB involves a reversible modification at the active site. To further test the conversion of p-NPGB, AEH was incubated with p-NPGB, and the formation ofp-NP was followed by stopped-flow spectroscopy. The reaction with p-NPGB followed a biphasic time course (Fig.2 B), consisting of an initial burst followed by a phase that corresponds to the steady-state hydrolysis. The formation of the acyl-enzyme intermediate was faster than its hydrolysis, resulting in an accumulation of the acyl-enzyme and the burst of p-NP, which is in agreement with what is expected for an active site-directed covalent inhibitor. Subsequently, in the steady-state phase, the acyl-enzyme complex was slowly hydrolyzed with ak cat of 1.3 ± 0.6 × 10−3 s−1. The steady-state rate of the conversion of p-NPGB within the concentration range of 0.1 to 1 mm p-NPGB was constant (data not shown), indicating that the K mfor p-NPGB is <0.1 mm. Therefore, the burst at 1 mm p-NPGB can be directly related to the number of active sites. The burst was measured in duplicate with two different enzyme concentrations and was found to correspond to 2.7 ± 0.7 μm released product with 1.32 μmenzyme and 1.1 ± 0.2 μm released product with 0.66 μm enzyme. In view of the subunit composition, this indicates that each subunit has one active site. The slow conversion of the acyl-enzyme intermediate during reaction of p-NPGB made it possible to covalently label the enzyme (Fig. 3). AEH was incubated with excess p-NPGB, and the covalent form was trapped by the addition of acid and subsequently fragmented with CNBr. Twenty peptide fragments in which the methionines had been modified to homoserine lactone were generated, varying in mass from 0.102 to 20.9 kDa. The elution pattern of the peptide mixture obtained from labeled AEH showed a few different peaks compared with the control (Fig.4). These peaks were individually collected and analyzed by ES/MS. The peak indicated as the control in the HPLC elution pattern (Fig. 4) corresponded to the fragment562GGYELPVSM570 (903.4 Da), indicated by its singly, (M + H)+, and doubly, (M + 2H)2+, charged peak in the mass spectrum, m/z 904.4 and 452.6, respectively. This fragment had the same mass when isolated from unlabeled or p-NPGB-labeled protein (Fig.5, A and B). Peak 1 could not be assigned to an expected CNBr fragment and is likely the result of incomplete digestion. ES/MS analysis of peak 3 showed a mixture of peptides, and the major component of the mixture did not change upon labeling. The peptide eluting in peak 2 was identified as CNBr fragment 202TGSSYEGFTVVM213(1228.6 Da), of which m/z 1229.5, (M + H)+, andm/z 615.6, (M + 2H)2+, were present in the ES/MS analysis of the unlabeled protein (Fig. 5 C). When isolated from protein that was preincubated with p-NPGB, a peptide was found at this position with a mass of 1390 Da, indicated by the peaks with m/z 1390.8, (M + H)+, andm/z 696.0, (M + 2H)2+ (Fig. 5 D). This mass is in agreement with the fragment of 1228.6 Da plus the guanidino benzoate label (161 Da; Fig. 3), indicating that the fragment that harbors the potential active site serine was labeled byp-NPGB. The increase in absorbance of the peptide after labeling is in agreement with the attachment of an aromatic group. The presence of the (M + H)+ ion at m/z 1229.5 in the spectrum of the labeled peptide fragment is probably due to some fragmentation in the orifice skimmer region of the mass spectrometer, resulting in loss of the charged label.Figure 4HPLC elution pattern of CNBr-peptide fragments of labeled (solid line) and unlabeled (dotted line) AEH. The peak indicated as control and peaks 1–3 were analyzed by mass spectrometry.View Large Image Figure ViewerDownload (PPT)Figure 5ES/MS spectra of the CNBr peptide fragments generated from AEH after labeling with p-NPGB.Shown are MS spectra of peptide Gly562-Met570(control peptide, A and B) and peptide Thr202-Met213 (C and D). The peptides were obtained from unlabeled enzyme (A andC) or from AEH preincubated with p-NPGB (B and D). cps, counts/second;amu, atomic mass unit.View Large Image Figure ViewerDownload (PPT) To determine which serine (204 or 205) of fragment202TGSSYEGFTVVM213 was modified byp-NPGB, the labeled peptide was analyzed by ES/tandem mass spectrometry using product ion scan to obtain the significant fragments. The expected b+ fragments for the peptides labeled at either 204 or 205 were calculated (Fig.6 A) and compared with the data. The product ion scan of the precursor ion m/z 1229.5, (M + H)+, of the unlabeled peptide displayed most of the possible b+ fragments, together with the precursor ion itself (Fig. 6, A and B). The product ion scan of the (M + 2H)2+ ion at m/z 695.9 of the labeled peptide showed an increase in the masses by 161 Da of the b+ fragments starting at b4, compared with the unlabeled protein (Fig. 6, A and C). The same increase in mass was found only for the detected y fragments 2Amide bond cleavage yields b and y ions containing the N or C terminus, respectively. y9+ (hsl-Ser205) to y11+ (hsl-Gly203) of the labeled peptide compared with the unlabeled peptide (data not shown).
