Acetobacter turbidans α-Amino Acid Ester Hydrolase
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m511187200
ISSN1083-351X
AutoresT.R.M. Barends, Jolanda J. Polderman-Tijmes, Peter A. Jekel, Christopher Williams, Gjalt G. Wybenga, Dick B. Janssen, Bauke W. Dijkstra,
Tópico(s)Biochemical and Molecular Research
ResumoThe α-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of β-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product d-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which d-phenylglycine binds, and an additional space that is large enough to accommodate the β-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr112, which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr112 side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the β-lactam binding site, this could explain the increased β-lactam transferase activity of this mutant. The α-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of β-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product d-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which d-phenylglycine binds, and an additional space that is large enough to accommodate the β-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr112, which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr112 side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the β-lactam binding site, this could explain the increased β-lactam transferase activity of this mutant. Thirty years ago, several bacterial strains, such as Acetobacter turbidans and Xanthomonas citri, were identified that were able to efficiently produce semi-synthetic β-lactam antibiotics from β-lactam nuclei produced by fermentation, and synthetic acyl compounds with an α-amino group (1Takahashi T. Yamazaki Y. Kato K. Isona M. J. Am. Chem. Soc. 1972; 94: 4035-4037Crossref PubMed Scopus (60) Google Scholar). Important antibiotics with such acyl chains include cephalexin, cephadroxil, ampicillin, and amoxicillin. Given the difficulties in preparing such antibiotics by chemical means (2Bruggink A. Roos E.C. de Vroom E. Org. Process Res. Dev. 1998; 2: 128-133Crossref Scopus (289) Google Scholar), much effort has been put into harnessing the β-lactam antibiotic synthesizing activity of these bacteria for application in the industrial production of antibiotics. It appeared that this activity originated from enzymes preferentially hydrolyzing esters of α-amino acids, the α-amino acid ester hydrolases (AEHs) 2The abbreviations used are: AEH,α-amino acid ester hydrolase; d-PGM, d-phenylglycine methyl ester; WT, wild-type. 2The abbreviations used are: AEH,α-amino acid ester hydrolase; d-PGM, d-phenylglycine methyl ester; WT, wild-type. (3Takahashi T. Yamazaki Y. Kato K. Biochem. J. 1974; 137: 497-503Crossref PubMed Scopus (45) Google Scholar).Because of its potential usefulness in antibiotic synthesis, the AEH from A. turbidans has been studied extensively, and it was the first of its family for which the gene was cloned and overexpressed (4Polderman-Tijmes J.J. Jekel P.A. de Vries E.J. van Merode A.E.J. Floris R. van der Laan J.M. Sonke T. Janssen D.B. Appl. Env. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). The sequence showed a GXSYXG active site motif (4Polderman-Tijmes J.J. Jekel P.A. de Vries E.J. van Merode A.E.J. Floris R. van der Laan J.M. Sonke T. Janssen D.B. Appl. Env. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar), which is characteristic of serine hydrolases of the X-prolyl dipeptidyl aminopeptidase family (5Chich J.-F. Chapot-Chartier M.-P. Ribadeau-Dumas B. Gripon J.-C. FEBS Lett. 1992; 314: 139-142Crossref PubMed Scopus (29) Google Scholar). Labeling studies with a suicide inhibitor, sequence alignments, and site-directed mutagenesis identified a catalytic triad of Ser205, Asp338, and His370 in what was proposed to be a catalytic domain with an α/β-hydrolase fold (6Polderman-Tijmes J.J. Jekel P.A. Jeronimus-Stratingh C.M. Bruins A.P. van der Laan J.M. Sonke T. Janssen D.B. J. Biol. Chem. 2002; 277: 28474-28482Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).Recently, the crystal structure of the X. citri AEH was solved (7Barends T.R.M. Polderman-Tijmes J.J. Jekel P.A. Hensgens C.M.H. de Vries E.J. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 2003; 278: 23076-23084Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This enzyme shares 63% sequence identity with the A. turbidans AEH. The structure showed a tetrameric arrangement of monomers consisting of three domains each: an α/β-hydrolase domain at the N terminus, a helical cap domain, and a C-terminal jellyroll fold domain. The active site indeed contained a Ser-His-Asp catalytic triad, the constituents of which were found in their canonical positions in the α/β-hydrolase domain. Furthermore, a putative oxyanion hole was found in which a negative charge could be stabilized by a backbone amide and a tyrosine hydroxyl group. Similar folds and arrangements of active site residues were observed for the homologous cocaine esterase CocE (8Larsen 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 (102) Google Scholar) and Lactococcus lactis X-prolyl dipeptidyl aminopeptidase PepX (9Rigolet P. Mechin I. Delage M.-M. Chich J.-F. Struct. Fold. Des. 2002; 10: 1383-1394Abstract Full Text Full Text PDF Scopus (19) Google Scholar). A particularly striking feature of the Xanthomonas AEH active site was a cluster of three carboxylate residues, which are conserved among the AEHs and were proposed to bind the positively charged α-amino group of the substrate from which the AEHs draw their name (7Barends T.R.M. Polderman-Tijmes J.J. Jekel P.A. Hensgens C.M.H. de Vries E.J. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 2003; 278: 23076-23084Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).The coupling of acyl side chains to β-lactams by AEHs most likely proceeds through enzyme acylation at the catalytic serine residue by an acyl compound, followed by transfer of this acyl group to the amino group of an amino-β-lactam, through “aminolysis” of the acyl enzyme (3Takahashi T. Yamazaki Y. Kato K. Biochem. J. 1974; 137: 497-503Crossref PubMed Scopus (45) Google Scholar, 4Polderman-Tijmes J.J. Jekel P.A. de Vries E.J. van Merode A.E.J. Floris R. van der Laan J.M. Sonke T. Janssen D.B. Appl. Env. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar, 10Kato K. Agric. Biol. Chem. 1980; 44: 1083-1088Google Scholar, 11Nam D.H. Kim C. Ryu D.D.Y. Biotech. Bioeng. 1985; 27: 953-960Crossref PubMed Scopus (41) Google Scholar). However, the acyl enzyme can also be hydrolyzed, and the synthesized antibiotic can also serve as substrate and be cleaved, as indicated in Fig. 1. Thus, the hydrolysis of the ester and the product occur as undesired side reactions (3Takahashi T. Yamazaki Y. Kato K. Biochem. J. 1974; 137: 497-503Crossref PubMed Scopus (45) Google Scholar, 11Nam D.H. Kim C. Ryu D.D.Y. Biotech. Bioeng. 1985; 27: 953-960Crossref PubMed Scopus (41) Google Scholar, 12Blinkovsky A.M. Markaryan A.N. Enzyme Microb. Technol. 1993; 15: 965-973Crossref PubMed Scopus (35) Google Scholar, 13Fernandez-Lafuente R. Hernández-Jústiz O. Mateo C. Terreni M. Alonso J. Garcia-López J.L. Moreno M.A. Guisan J.M. J. Mol. Catal. B. Enz. 2001; 11: 633-638Crossref Scopus (24) Google Scholar). Therefore, the AEH-catalyzed synthesis of antibiotics requires a kinetically controlled scheme. In such a scheme, the enzyme is added to a mixture of the acyl donor, e.g. d-phenylglycine methyl ester, and the acyl acceptor, e.g. 6-aminopenicillanic acid. All three reactions (synthesis of the antibiotic, hydrolysis of the ester, and hydrolysis of the antibiotic) will occur simultaneously, but after a certain period of time, a maximum in the antibiotic concentration is reached, at which time the reaction is stopped and the product harvested. The kinetic parameters of the enzyme govern the maximum level of antibiotic accumulation PSmax in such a process (14Youshko M.I. Chilov G.G. Shcherbakova T.A. Svedas V.K. Biochim. Biophys. Acta. 2002; 1599: 134-140Crossref PubMed Scopus (60) Google Scholar). Of crucial importance is the ratio of the specificity constants of the enzyme toward the acyl donor and the antibiotic, α, which is defined as (kcat/Km)product/(kcat/Km)acyl donor. For favorable antibiotic yield, α needs to be as small as possible. Another important factor is the ratio of the rates of synthesis and hydrolysis, measured as the ratio of the initial rates, Vs/Vhini. At a certain concentration of nucleophile, this ratio indicates the tendency of the acyl enzyme to undergo aminolysis rather than hydrolysis. For optimal antibiotic yield, Vs/Vhini needs to be maximal (14Youshko M.I. Chilov G.G. Shcherbakova T.A. Svedas V.K. Biochim. Biophys. Acta. 2002; 1599: 134-140Crossref PubMed Scopus (60) Google Scholar, 15Alkema, W. B. L. (2002) Faculty of Mathematics and Natural Sciences, Ph.D. thesis, pp. 142, University of Groningen, GroningenGoogle Scholar). Kinetic work has shown that mutation of Tyr206 to alanine decreases the unwanted hydrolytic activity toward cephalexin in the Acetobacter enzyme (6Polderman-Tijmes J.J. Jekel P.A. Jeronimus-Stratingh C.M. Bruins A.P. van der Laan J.M. Sonke T. Janssen D.B. J. Biol. Chem. 2002; 277: 28474-28482Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The corresponding residue in the Xanthomonas AEH was proposed to contribute to transition state stabilization with its main chain atoms (7Barends T.R.M. Polderman-Tijmes J.J. Jekel P.A. Hensgens C.M.H. de Vries E.J. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 2003; 278: 23076-23084Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The Y206A mutant of Acetobacter AEH was further investigated in terms of its usefulness as a biocatalyst for antibiotic production (16van der Laan, J. M., Polderman-Tijmes, J. J., and Barends, T. R. M. (2002) International Patent Application WO 02/086111 A2Google Scholar).The present work reports the structure of the AEH from A. turbidans, aimed at understanding the precise mode of action of this enzyme. In addition to the native structure, the structures of the complex with d-phenylglycine, the Y206A mutant, and the inactive S205A mutant complexed with the antibiotic ampicillin are reported, as are the effects of the Y206A mutation on the kinetic parameters α, Vs/Vhini, and PSmax. The structures give insight into the catalytic residues, substrate binding, and the effects of mutations. In particular, the structure of the Y206A mutant helps to explain the higher ratio of the rates of synthesis and hydrolysis that this mutant displays.EXPERIMENTAL PROCEDURESProtein Production—Plasmids, bacterial strains, growth conditions, and purification methods for the production of native, S205A, and Y206A A. turbidans AEH with a C-terminal myc epitope and His tag were as described in Ref. 6Polderman-Tijmes J.J. Jekel P.A. Jeronimus-Stratingh C.M. Bruins A.P. van der Laan J.M. Sonke T. Janssen D.B. J. Biol. Chem. 2002; 277: 28474-28482Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar. Briefly, proteins were produced in Escherichia coli TOP10 cells carrying constructs derived from pBAD. Cells were grown at 14 °C for 4 days in LB medium with 100 μg/ml ampicillin and 0.01% (w/v) arabinose for induction. After harvesting and washing, cells were either sonicated or passed through a French press and a clear lysate was prepared by centrifugation. AEH and mutant AEHs were purified from this lysate by metal ion affinity chromatography using nickel-nitrilotriacetic acid-agarose (Qiagen). A stepwise gradient of 50-200 mm imidazole in 150 mm NaCl, 50 mm sodium phosphate, pH 7.4, was used for elution. Pure protein eluted around 75-100 mm imidazole. Subsequently, the protein was desalted using gel filtration. Determination of the oligomeric state was carried out by gel filtration on a Superdex 200 column equilibrated with 50 mm sodium phosphate buffer, pH 6.2, containing 200 mm sodium chloride. Elution volumes were calibrated using Bio-Rad gel filtration markers.Determination of Kinetic Parameters—For the determination of enzyme behavior in a cephalexin synthesis reaction, the enzymes were incubated with 15 mm d-phenylglycine methyl ester (d-PGM) and 30 mm β-lactam nucleus 7-amino-desacetoxycephalosporanic acid in 50 mm Na-phosphate buffer, pH 6.2, at 30 °C. Kinetic parameters for cephalexin hydrolysis were previously reported in Ref. 6Polderman-Tijmes J.J. Jekel P.A. Jeronimus-Stratingh C.M. Bruins A.P. van der Laan J.M. Sonke T. Janssen D.B. J. Biol. Chem. 2002; 277: 28474-28482Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar and are given here for reference. The synthesis of cephalexin and the hydrolysis of d-PGM were followed by high performance liquid chromatography as described before (4Polderman-Tijmes J.J. Jekel P.A. de Vries E.J. van Merode A.E.J. Floris R. van der Laan J.M. Sonke T. Janssen D.B. Appl. Env. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). Enzymes were used at a concentration that catalyzed the hydrolysis of d-PGM with an initial rate of 0.20 to 0.30 mm/min. To obtain the rate of enzymatic d-phenylglycine production, the observed rate of d-phenylglycine production was corrected for the first-order chemical hydrolysis of d-PGM. The relative rate of antibiotic formation versus hydrolysis of d-PGM (Vs/Vhini) was determined from the initial slope of antibiotic formation divided by the rate of enzymatic formation of the hydrolysis product (d-phenylglycine). The rates were measured at less than 10% conversion of d-PGM to minimize the influence of product hydrolysis. The kinetic parameters Km and kcat were calculated using nonlinear regression fitting (Scientist, Micromath) with Michaelis-Menten and substrate inhibition kinetics. The calculated parameters are given with their standard deviations. The maximum product concentration (Vs/Vhini) was determined by following the concentrations of cephalexin and d-phenylglycine over time, until the product concentration started to decrease. All measurements were at least performed in duplicate.Crystal Preparation—His-tagged native and mutant AEHs were concentrated to 5 mg/ml by ultrafiltration. WT A. turbidans AEH and the Y206A mutant were crystallized by mixing 3 μl of protein solution with 3 μl of 15-17% PEG 4000, 0.2 m ammonium acetate, 0.1 m sodium acetate buffer, pH 4.6, followed by equilibration against this PEG solution. Prism-shaped crystals of WT protein of maximum dimensions 0.2 × 0.05 × 0.05 mm were obtained in 1 week. Plate-shaped crystals (0.2 × 0.2 × 0.01 mm) of the Y206A mutant were obtained in 4 days. In an attempt to obtain a complex with a substrate, WT crystals were transferred to mother liquor containing 10 mg/ml ampicillin for 30 min after which they were cryoprotected by soaking for a few seconds in the same solution containing 25% glycerol, and cryocooled in liquid nitrogen. Y206A crystals were subjected to the same procedure, substituting d-phenylglycine methyl ester for ampicillin. Crystals of a S205A mutant-ampicillin complex were grown by mixing 3 μl of protein solution with 3 μl of a reservoir solution containing 0.3 m sodium citrate buffer, pH 5.6, 13-14% PEG 4000, and 10 mm sodium ampicillin, followed by equilibration in hanging drops against 500 μl of this latter solution for 3-4 days. Rhombic dodecahedra with a size of 0.3 × 0.3 × 0.3 mm were harvested and cryocooled after soaking for 30 s in reservoir solution containing 26% glycerol. WT·d-phenylglycine complex crystals were grown in the same way, substituting WT protein for the mutant and d-phenylglycine for ampicillin. All crystallization and soaking experiments were carried out at room temperature.Data Collection and Structure Solution—Useful diffraction data from WT, WT·d-phenylglycine, and Y206A crystals were collected to 2.0-, 3.3-, and 2.8-Å resolution, respectively, on beam line ID14-4 at the ESRF in Grenoble, France. Diffraction data from crystals of the S205A mutant co-crystallized with ampicillin were collected to 2.2-Å resolution on the BW7B beam line of the EMBL outstation at the DESY synchrotron in Hamburg, Germany. All data were processed with DENZO and SCALE-PACK (17Otwinowksi Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar). The structure of the A. turbidans AEH was solved by molecular replacement, using the program AMoRe (18Navaza J. Acta Crystallogr. Sect. A Crystallogr. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) and the previously determined tetrameric structure of the X. citri AEH as a search model. The structure determination of the WT protein was hampered by twinning in combination with pseudo-crystallographic translation symmetry, and the structure elucidation process followed with this protein is further described in Ref. 19Barends T.R.M. Dijkstra B.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2237-2241Crossref PubMed Scopus (20) Google Scholar. Briefly, a high twinning fraction was observed, which made it impossible to detwin the data mathematically. Therefore, the twinning was idealized by averaging intensities of twin-related reflections (20Yeates T.O. Methods Enzymol. 1997; 276: 344-358Crossref PubMed Scopus (356) Google Scholar) and the structure of the one twin domain, solved by molecular replacement, was used to obtain detwinned intensities for refinement. It should be noted, that because the measured data are relatively incomplete (85.5% completeness), not all twin-related reflection pairs could be averaged for lack of availability of one of the reflections. After averaging and detwinning, this leads to the availability of only 75% of all possible reflections for refinement. However, this number of available reflections corresponds to the number of reflections that a 100% complete dataset of 2.2-Å resolution from this crystal would have, thus effectively limiting the resolution to ∼2.2 Å. The twinning also results in the loss of independence of twin-related reflections, lowering the observation/parameter ratio further. However, there are 16 AEH monomers in the asymmetric unit (four AEH tetramers), enabling the use of tight 16-fold non-crystallographic symmetry restraints, which ensures that the refinement problem is sufficiently well determined.Final models of all proteins were obtained through iterative cycles of refinement with Refmac 5.0 (21Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar), combined with rebuilding in Xfit (22McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar). During refinement of the Y206A mutant structure, 8-fold non-crystallographic symmetry restraints were employed. Data collection and refinement parameters are given in Table 1.TABLE 1Data collection and refinement statisticsData collectionsWTWT-d-phenylglycineS205A-ampicillinY206AStatisticsProtein Data Bank code2B9V2B4K1NX91RYYWavelength (Å)0.9330.9340.84630.9393Space group and cell dimensionsP21, a = 98.4, b = 275.6, c = 197.0 Å, β = 90.1°I23, a = b = c = 341.0 Å, α = β = γ = 90°I23, a = b = c = 341.8 Å, α = β = γ = 90°P21, a = 91.6, b = 177.5, c = 170.0 Å, β = 91.0°Resolution range (Å)40-2.040-3.0aDue to the high merging R-factor at 3.0-Å resolution, only data to 3.3-Å resolution were used for refinement.99-2.240-2.8No. of unique reflections598,744121,384322,911134,932Completeness (%)85.5 (67.8)93.2 (91.5)97.2 (84.3)99.4 (99.6)Redundancy2.82.64.53.2Rsym0.091 (0.341)0.186 (0.917)0.081 (0.322)0.066 (0.184)I/σ6.6 (1.5)7.1 (2.8)14.3 (3.7)17.2 (6.9)Refinement statisticsResidue range in refinement (Å)40-2.0bThe data used for the refinement of the WT structure are only 75% complete due to collection geometry and detwinning; however, the number of reflections used in the refinement of this structure corresponds to the number of reflections in a 100% complete dataset to 2.2-Å resolution.15-3.315-2.240-2.8No. reflections in refinement504,37991,758304,675125,967Protein atoms77,07219,53619,53239,016Water molecules3,65462,2290Ligand atomsd-Phenylglycine, 33 (3 molecules); glycerol, 18 (3 molecules)Ampicillin, 96 (4 molecules); glycerol, 24 (4 molecules)0R-factor0.1990.2580.1660.253Rfree0.2350.2890.1840.272Root mean square deviations from ideal geometryBonds (Å)0.0080.0090.0070.009Angles (°)1.71.01.10.8Ramachandran plotMost favored87.0%82.7%86.0%84.1%Additional allowed12.6%17.1%13.6%15.4%Disallowed (these include catalytic and structurally important residues)0.3%0.2%0.4%0.4%a Due to the high merging R-factor at 3.0-Å resolution, only data to 3.3-Å resolution were used for refinement.b The data used for the refinement of the WT structure are only 75% complete due to collection geometry and detwinning; however, the number of reflections used in the refinement of this structure corresponds to the number of reflections in a 100% complete dataset to 2.2-Å resolution. Open table in a new tab RESULTS AND DISCUSSIONNative Structure: Oligomeric State—The structures of the WT, WT·d-phenylglycine, Y206A, and S205A AEH were successfully determined by x-ray crystallography. Despite different space groups and crystallization conditions, all structures showed the same tetrameric arrangement of monomers (Fig. 2a), which was also observed for the X. citri AEH (7Barends T.R.M. Polderman-Tijmes J.J. Jekel P.A. Hensgens C.M.H. de Vries E.J. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 2003; 278: 23076-23084Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Gel filtration experiments with the WT protein and the S205A mutant indicated the presence of exclusively tetramers in solution, reinforcing the notion that the crystallographically observed tetramer represents the physiological oligomerization state.FIGURE 2a, stereo figure of the A. turbidans α-amino acid ester hydrolase tetramer. A surface representation is shown, in which each monomer is individually colored. b, stereo figure of the A. turbidans α-amino acid ester hydrolase monomer. The arm and α/β-hydrolase domains are shown in green, the cap domain in orange, and the jellyroll domain in blue. The side chain of the active site Ser205 is shown in space filling representation. Helices αC-αF and strands β4-β7 are labeled. The figure was prepared using PYMOL (DeLano Scientific) and Molscript (30Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Acetobacter AEH tetramer (Fig. 2a) has the shape of a hollow sphere with a diameter of about 100 Å. The central cavity can only be reached through two diametrically opposed entrances that are each ∼15 Å wide. Because the active sites are found on the inside of the sphere, this tetrameric oligomerization state would restrict the AEHs to converting small compounds.The native structure of the Acetobacter AEH monomer (Fig. 2b) resembles that of the Xanthomonas AEH monomer. It can be superimposed with Xanthomonas AEH to within a root mean square difference of Cα positions of 4.5 Å. Like the Xanthomonas enzyme, it includes an N-terminal arm, an α/β-hydrolase domain with a helical cap domain, and a C-terminal jellyroll fold domain. The N-terminal arm consists of 24 residues that form an elongated polypeptide that interacts with the surface of another monomer. Residues 62-73, in the middle of the arm, could not be identified in the electron density map. These residues span the gap between the two monomers held together by the arm, and may thus be expected to be mobile, because they make no non-bonded contacts with either monomer. However, their approximate positions can be inferred from the short distance between the beginning and end of this gap and their better defined positions in the S205A mutant structure.The arm is followed by a typical α/β-hydrolase fold domain, consisting of a highly twisted β-sheet flanked on both sides by α-helices (23Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1830) Google Scholar, 24Heikinheimo P. Goldman A. Jeffries C. Ollis D.L. Struct. Fold Des. 1999; 7: R141-R146Abstract Full Text Full Text PDF Scopus (219) Google Scholar, 25Nardini M. Dijkstra B.W. Curr. Opin. Struct. Biol. 1999; 9: 732-737Crossref PubMed Scopus (668) Google Scholar). Apart from the second one, all strands in the sheet are parallel. Adopting the nomenclature in Ref. 23Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1830) Google Scholar, the catalytic residues Ser205, His370, and Asp338 are found at their canonical positions on the loops between strand β5 and helix αC, between β8 and αF, and between β7 and αE, respectively. Compared with the classical α/β-hydrolase fold, which has eight β-strands in the central sheet, AEH displays two additional β-strands at the C terminus of the domain. Furthermore, several insertions are seen, most notably a predominantly helical cap domain between β6 and αD. Another insertion between β3 and αA contains the conserved Tyr112, which is in a position to contribute with its hydroxyl group to oxyanion stabilization during the reaction. The C-terminal domain adopts a jellyroll fold with several insertions, one of which contains a helix that approaches the active site as in the X. citri AEH.Active Site Structure—The active site is found at the interface of the α/β-hydrolase, cap, and jellyroll domains. The catalytic Ser205 is located on the so-called nucleophile elbow between β5 and αC and has unfavorable main chain dihedrals as is usual in α/β-hydrolase family members (25Nardini M. Dijkstra B.W. Curr. Opin. Struct. Biol. 1999; 9: 732-737Crossref PubMed Scopus (668) Google Scholar). It is closely approached by His370, which in turn is hydrogen bonded to Asp338. Thus, a canonical Ser-His-Asp catalytic triad is formed. The backbone amide of Tyr206, which directly follows the catalytic serine in the sequence, and the phenolic OH of the Tyr112 side chain form an oxyanion hole similar to that observed in X. citri AEH, CocE, and PepX (7Barends T.R.M. Polderman-Tijmes J.J. Jekel P.A. Hensgens C.M.H. de Vries E.J. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 2003; 278: 23076-23084Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 8Larsen 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 (102) Google Scholar, 9Rigolet P. Mechin I. Delage M.-M. Chich J.-F. Struct. Fold. Des. 2002; 10: 1383-1394Abstract Full Text Full Text PDF Scopus (19) Google Scholar). As in these proteins, Tyr112 makes stacking-like interactions with the Pro111 ring, as does Tyr206 on the other side of the Pro111 side chain (figure 3a). The Tyr112 side chain is further restrained by a hydrogen bond with the Tyr253 side chain (not shown).FIGURE 3a, stereo figure showing the active site residues of A. turbidans AEH. The catalytic triad and the residues forming the oxyanion hole are shown in stick representation. An asterisk indicates the expected position of the oxyanion hole. The 2Fo - Fc electron density map is overlaid on the structure at a 1.0-σ contour level. b, stereo figure, showing (Fo - Fc) difference electron density calculated with the d-phenylglycine co-crystal data prior to including d-phenylglycine in the model, overlaid on the refined d-phenylglycine complex structure. The ammonium group of d-phenylglycine is in close proximity to the carboxylate cluster. c, stereo figure comparing the active site residues of WT (gray) and Y206A (blue) AEH. Transparent spheres indicate the van der Waals radii of the atoms of the interacting Tyr112 and Asn257 residues in the Y206A structure. The changes upon the mutation of Tyr206 to Ala are indicated with numbers: 1, mutation of Tyr206 to Ala; 2, loss of the hydrogen bond with Tyr160; 3, movement of the Gln257 side chain; 4, van der Waals interactions of Gln257 side chain with Tyr112 side chain; 5, movement of Tyr112 side chain toward oxyanion hole; 6, possible hydrogen bond between Tyr112 Oη and Ser205 Oγ. The figure was prepared using Xfit (22McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar), BobScript (31Esnouf R.M. J. Mol. Graph. 1997; 15: 133-138Google Scholar), and Raster3D (32Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar).View Large
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