FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019547185> ?p ?o ?g. }

• W2019547185 endingPage "19904" @default.
• W2019547185 startingPage "19894" @default.
• W2019547185 abstract "BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (λmax is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (λmax is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (λmax is 506 nm) with a similar rate, 1/τ ∼ 500 s-1. The crystal structure of the S112A:HOPDA complex at 1.8-Å resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7Å away. BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (λmax is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (λmax is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (λmax is 506 nm) with a similar rate, 1/τ ∼ 500 s-1. The crystal structure of the S112A:HOPDA complex at 1.8-Å resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7Å away. Bacteria use meta-cleavage pathways to degrade a large variety of aromatic compounds, and some alicyclic compounds, such as steroids (1Vaillancourt F.H. Bolin J.T. Eltis L.D. Crit. Rev. Biochem. Mol. Biol. 2006; 41: 241-267Crossref PubMed Scopus (301) Google Scholar). Although each pathway has its own substrate specificity, they all employ the same underlying logic: vicinal dihydroxylation of an aromatic ring enables dioxygenase-catalyzed extradiol (or meta) ring opening. The resulting meta-cleavage product (MCP) 3The abbreviations used are: MCP, meta-cleavage product; HOPDA, 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid; HPD, 2-hydroxy-2,4-pentadienoic acid; Bph, biphenyl; PCB, polychlorinated biphenyl. 3The abbreviations used are: MCP, meta-cleavage product; HOPDA, 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid; HPD, 2-hydroxy-2,4-pentadienoic acid; Bph, biphenyl; PCB, polychlorinated biphenyl. is degraded by an MCP hydrolase, which adds water across a carbon-carbon bond to generate a dienoate and a carboxylic acid (Fig. 1). Enzymes of the meta-cleavage pathway have been of interest because of their roles in biodegradation. For instance, the biphenyl (Bph) catabolic pathway transforms a number of polychlorinated biphenyls (PCBs). This process is limited by the inhibitory effects of PCBs or their chlorinated metabolites (2Seah S.Y.K. Labbé G. Nerdinger S. Johnson M.R. Snieckus V. Eltis L.D. J. Biol. Chem. 2000; 275: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 3Furukawa K. Trends Biotechnol. 2003; 21: 187-190Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). More recently, it was discovered that Mycobacterium tuberculosis catabolizes cholesterol via a meta-cleavage pathway (4Van der Geize R. Yam K. Heuser T. Wilbrink M.H. Hara H. Anderton M.C. Sim E. Dijkhuizen L. Davies J.E. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (406) Google Scholar) that is essential for pathogen survival in the macrophage (5Rengarajan J. Bloom B.R. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8327-8332Crossref PubMed Scopus (551) Google Scholar). A better understanding of the meta-cleavage pathway enzymes should accelerate the development of their potential for biocatalysis and biodegradation, as well as facilitate the design of novel therapeutics for the treatment of tuberculosis.MCP hydrolases, exemplified by BphD of the Bph pathway and HsaD of the cholesterol catabolic pathway, contain the canonical structural fold and Ser-His-Asp catalytic triad characteristic of the α/β-hydrolase enzyme superfamily (6Bugg T.D.H. Bioorg. Chem. 2004; 32: 367-375Crossref PubMed Scopus (56) Google Scholar, 7Heikinheimo P. Goldman A. Jeffries C. Ollis D.L. Structure. 1999; 7: 141-146Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 8Holmquist M. Curr. Prot. Pept. Sci. 2000; 1: 209-235Crossref PubMed Scopus (472) Google Scholar, 9Ollis 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. Verscheuren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1830) Google Scholar). This triad is associated with the classical hydrolytic mechanism typified by the serine proteases, in which the Asp-His dyad first activates Ser to nucleophilic attack at the substrate carbonyl, and subsequently activates water to release the resulting acyl-enzyme covalent intermediate. Although the catalytic Ser and Asp residues vary within the nucleophile-His-acid framework, all known α/β-hydrolase active sites contain the catalytic His and the oxyanion hole. The latter, which stabilizes the negative charge of the tetrahedral intermediate oxyanion, is created by the partial positive charges of backbone amide protons.The proposed mechanism of MCP hydrolases differs from that of other α/β-hydrolases in two important respects: (i) it invokes an enol-keto tautomerization prior to hydrolysis (Fig. 1) (10Henderson I.M.J. Bugg T.D.H. Biochemistry. 1997; 36: 12252-12258Crossref PubMed Scopus (38) Google Scholar, 11Lam W.W.Y. Bugg T.D.H. Biochemistry. 1997; 36: 12242-12251Crossref PubMed Scopus (48) Google Scholar) and (ii) it asserts hydrolysis via a gem-diol intermediate rather than an acyl-enzyme (12Fleming S.M. Robertson T.A. Langley G.J. Bugg T.D.H. Biochemistry. 2000; 39: 1522-1531Crossref PubMed Scopus (55) Google Scholar, 13Li J.J. Li C. Blindauer C.A. Bugg T.D.H. Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar, 14Speare D.M. Fleming S.M. Beckett M.N. Li J.J. Bugg T.D.H. Org. Biomol. Chem. 2004; 2: 2942-2950Crossref PubMed Google Scholar, 15Speare D.M. Olf P. Bugg T.D.H. Chem. Commun. 2002; : 2304-2305Crossref PubMed Google Scholar, 16Li J.J. Bugg T.D.H. Org. Biomol. Chem. 2007; 5: 507-513Crossref PubMed Google Scholar). The two best studied MCP hydrolases are BphD from Burkholderia xenovorans LB400, which hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD), and MhpC involved in phenylpropionic acid degradation. Kinetic and structural studies of both enzymes have indicated that the catalytic His may mediate tautomerization, as well as help catalyze hydrolysis (13Li J.J. Li C. Blindauer C.A. Bugg T.D.H. Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar, 17Dunn G. Montgomery M.G. Mohammed F. Coker A. Cooper J.B. Robertson T. Garcia J.L. Bugg T.D.H. Wood S.P. J. Mol. Biol. 2005; 346: 253-265Crossref PubMed Scopus (51) Google Scholar, 18Li C. Montgomery M.G. Mohammed F. Li J.J. Wood S.P. Bugg T.D.H. J. Mol. Biol. 2005; 346: 241-251Crossref PubMed Scopus (34) Google Scholar). For instance, stopped-flow studies showed that substitution of the catalytic histidine decreased the rate of a process proposed to represent tautomerization in MhpC (18Li C. Montgomery M.G. Mohammed F. Li J.J. Wood S.P. Bugg T.D.H. J. Mol. Biol. 2005; 346: 241-251Crossref PubMed Scopus (34) Google Scholar) and a polyhistidine-tagged BphD (Ht-BphD) (13Li J.J. Li C. Blindauer C.A. Bugg T.D.H. Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar). In the crystal structure of an MhpC:substrate analog binary complex, the catalytic His was 3.2 Å from the C2 carbonyl and 3.9 Å from C5 of the analog (17Dunn G. Montgomery M.G. Mohammed F. Coker A. Cooper J.B. Robertson T. Garcia J.L. Bugg T.D.H. Wood S.P. J. Mol. Biol. 2005; 346: 253-265Crossref PubMed Scopus (51) Google Scholar), consistent with histidine's role in abstracting a proton from the C2 hydroxyl and protonating C5. More convincingly, the crystal structure of the S112C variant of BphD incubated with HOPDA revealed a complex with the product HPD and an interaction between the catalytic His-265 and the 2-hydroxy/oxo substituent of the dienoate, consistent with a general base role in substrate tautomerization (19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar). Protonation by His-265 at HOPDA C5 was deemed possible because of the observed conformational flexibility of the former, allowing us to predict a His-265-to-C5 distance as short as 2.9 Å.A complicating factor in evaluating the catalytic role of His-265 is that the proposed keto intermediate has not been directly observed. Using single turnover ([E] > [S]) stopped-flow analysis, we recently identified an intermediate possessing an electronic absorption maximum that is red-shifted (λmax is 492 nm) from that of the free HOPDA enolate (λmax is 434 nm) (19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar). Two kinetic models were proposed, differing in their assignment of the red-shifted intermediate (E:SRed) to either the enolate (E:Se) or keto (E:Sk) tautomer of enzyme-bound HOPDA. A priori, it is more logical to assign the red-shifted species to E:Se as ketonization disrupts the conjugated system of the HOPDA enolate. However, electronic transition energies of small molecule ligands may be greatly perturbed in a protein environment (20D'Ordine R.L. Tonge P.J. Carey P.R. Anderson V.E. Biochemistry. 1994; 33: 12635-12643Crossref PubMed Scopus (51) Google Scholar, 21Tian F. Debler E.W. Millar D.P. Deniz A.A. Wilson I.A. Schultz P.G. Angew. Chem. Int. Ed. Engl. 2006; 45: 7763-7765Crossref PubMed Scopus (16) Google Scholar, 22Sekharan S. Sugihara M. Buss V. Angew. Chem., Int. Ed. Engl. 2007; 46: 269-271Crossref PubMed Scopus (80) Google Scholar) and assignment of E:SRed to an E:Sk species is more consistent with additional kinetic observations. First, under single turnover conditions, the decay of E:SRed was coupled to HPD formation, consistent with direct transformation of E:Sk to HPD. Second, increased solvent viscosity slowed a relaxation assigned to tautomerization in the E:Se model, and to diffusive HPD release in the E:Sk model. Because tautomerization is intramolecular and hence should not be affected by solvent viscosity, the E:Sk model provides a more reasonable interpretation of the data. Finally, the apparently ordered product release could be more readily explained via the E:Sk model. Identifying the E:SRed intermediate observed in BphD, therefore, represents an important step toward elucidating the mechanism of MCP hydrolases.Herein, we describe kinetic and structural studies of alanine substitutions of the active site catalytic triad residues Ser-112 and His-265 of BphD. Stopped-flow spectrophotometry of the variants revealed the catalytic contribution of each residue based on the accumulation of different intermediates, such as E:SRed. In parallel, substrate complexes of the BphD variants were characterized at high resolution by x-ray crystallography. Monitoring the ability of enzyme variants to catalyze tautomerization of HPD provided further insight into the catalytic roles of the substituted residues in the tautomeric half-reaction. Implications for catalysis in MCP hydrolases are discussed.MATERIALS AND METHODSChemicals—HOPDA was enzymatically generated from 2,3-dihydroxybiphenyl (DHB) using DHB dioxygenase (DHBD) as previously described (19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar). The preparation of DHB has been described (23Nerdinger S. Kendall C. Marchhart R. Riebel P. Johnson M.R. Yin C.F. Eltis L.D. Snieckus V. Chem. Commun. 1999; : 2259-2260Crossref Scopus (28) Google Scholar). HPD was generated together with benzoic acid by BphDLB400-catalyzed hydrolysis of HOPDA. Upon reaction completion (monitored by absorbance at 434 nm), the solution was acidified to pH ∼ 3 with 2 n HCl, extracted 3 times with 0.3 volumes of ethyl acetate, dried over anhydrous MgSO4 and rotary evaporated to dryness. All other chemicals were of analytical grade.Mutagenesis, Protein Expression, and Purification—BphD from B. xenovorans LB400 was produced and purified as previously described (19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar, 24Seah S.Y.K. Terracina G. Bolin J.T. Riebel P. Snieckus V. Eltis L.D. J. Biol. Chem. 1998; 273: 22943-22949Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Ser-112 of BphD was substituted with alanine (S112A) using the Transformer site-directed mutagenesis method (Clontech Laboratories, Palo Alto, CA). Briefly, the S112A mutagenic primer (primer S112A: 5′-GCGCCCCCCATGGCGTTGCCGACCAG-3′) and a second mutagenic selection primer to remove an EcoRI site (primer ODM: 5′-AGCTCGAATTGGTAATCATGG-3′) were mixed with pSS184, the pEMBL18 vector carrying bphD (24Seah S.Y.K. Terracina G. Bolin J.T. Riebel P. Snieckus V. Eltis L.D. J. Biol. Chem. 1998; 273: 22943-22949Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). After second strand synthesis and ligation using T4 DNA polymerase and T4 DNA ligase (GE Healthcare, Uppsala, Sweden), respectively, the DNA was digested with EcoRI to linearize wild-type plasmid, and transformed into Escherichia coli BMH 71-18 mutS. Plasmid isolated from this first transformation was subjected to a second round of EcoRI digestion and transformation, from which pSS184SA, carrying the mutated gene, was isolated. The mutated gene was cloned into pVLT31 using XbaI and HindIII restriction sites, and the resulting plasmid, pSS314SA, was used for protein production. Substitution of His-265 with alanine was performed using a 5′-phosphorylated primer (H265A: CTCCAAGTGCGGCGCTTGGGCGCAATGG-3′) and the QuikChange multi site-directed mutagenesis kit (Stratagene, La Jolla, CA). Genes encoding the single (H265A) and double (S112A/H265A) variants were generated using pSS184 and pSS184SA, respectively, yielding pSS184HA and pSS184SAHA. These constructs were used directly for protein production. The nucleotide sequences of variants were confirmed using an ABI 373 Stretch (Applied Biosystems, Foster City, CA) and Big-Dye v3.1 terminators.Enzyme Activity Measurements—All enzyme kinetic experiments were performed using potassium phosphate buffer, I = 0.1 m, pH 7.5, at 25 °C. Steady-state enzyme activities were obtained by monitoring the decrease in absorbance at 434 nm of the HOPDA enolate versus time using a Varian Cary 5000 spectrophotometer (Varian Canada, Mississauga, ON, Canada). The latter was equipped with a thermostatted cuvette holder maintained at 25.0 ± 0.5 °C, controlled by Cary WinUV software version 3.00. To measure enzyme activity toward HPD, a small volume (<0.5% v/v) of HPD/benzoic acid in ethanol was added to a buffered solution, and absorbance at 270 nm was monitored before and after addition of enzyme.The half-life of the S112A:HOPDA complex was determined by mixing 25 μm S112A and 5 μm HOPDA, and recording the absorption spectrum at 0.5-1-h intervals. The absorbance at 506 nm was plotted against time, and described by a first order decay using Excel (Microsoft, Redmond, WA).Stopped-flow Spectrophotometry—Experiments were conducted using an SX.18MV stopped-flow reaction analyzer (Applied Photophysics Ltd., Leatherhead, UK) equipped with a photodiode array detector. The drive syringe chamber and optical cell were maintained at 25 °C by a circulating water bath. Multiple wavelength data from the time courses of single turnover experiments were acquired using the Xscan software (Applied Photophysics Ltd.) and exported to Excel where replicate measurements from at least three shots were averaged. To obtain more data at earlier time points, a single wavelength was monitored using the SX18MV software. The data from at least three shots were averaged and equations for single or double exponentials were fit using the same software to obtain reciprocal relaxation times and amplitudes. Good fits were characterized by random variation in the fit residuals.Crystallization of BphD and Preparation of Substrate Complexes—A 1.6-Å resolution structure of BphD was previously determined using crystals grown from a solution containing 1.6 m ammonium sulfate (19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar, 24Seah S.Y.K. Terracina G. Bolin J.T. Riebel P. Snieckus V. Eltis L.D. J. Biol. Chem. 1998; 273: 22943-22949Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These crystals had space group P64 with a = 135.0 Å, c = 66.7 Å, and a sulfate ion bound in the active site. The failure of many attempts to prepare crystalline complexes by incubation of crystals with substrates suggested sulfate binding or the crystal form restricts formation of E:S complexes leading us to seek alternative crystallization procedures.New protocols were established for both wild type and variant enzymes using both commercial (Hampton Research) and non-commercial sodium malonate grid screens and the vapor diffusion method. Sitting drops (1-μl each of reservoir and protein solutions) were equilibrated at 20 °C against 500- or 1000-μl reservoirs. The protein sample contained 9-28 mg/ml protein in 20 mm HEPES pH 7.5. Crystals or crystalline precipitates were obtained between 1.5 and 2.4 m sodium malonate within a pH range of 6.0 and 7.0.Two crystal forms were obtained. Crystals of wild-type enzyme had space group P64, a = 135.2 Å, c = 66.3 Å, whereas crystals of the S112A and the S112A/H265A variants had space group I4122 with a = 117.3 Å, c = 87.3 Å. The hexagonal crystals are essentially isomorphous with the previously reported form obtained from ammonium sulfate and contain two protein monomers per asymmetric unit; the tetragonal form has one monomer in the asymmetric unit. Crystals of wild-type BphD grew over 12 weeks as hexagonal prisms in 2.0 m sodium malonate, pH 6.0 or 6.5. Crystals of S112A and S112A/H265A grew in 4-6 days as tetragonal rods in 1.9 m sodium malonate. The best crystals of the S112A variant were obtained at pH 7.0, whereas the best S112A/H265A crystals grew at pH 6.5.Crystals of HOPDA complexes were obtained by incubating crystals grown in the absence of HODPA in 30-60 μl of reservoir solution augmented with ∼15 mm HOPDA for 30-60 min at 20 °C.Diffraction Data Measurements and Processing—Crystals were prepared for flash-freezing by sequential transfer into solutions containing higher concentrations of sodium malonate. A mounting loop was used to transfer crystals from the growth drop into 60-μl volumes of reservoir solution, then into similar solutions containing 3.4 m and, finally, 3.7 m sodium malonate. The pH was held at the growth value; the incubation time was 3-6 s per step. After the last transfer crystals were flash-frozen by immersion into liquid nitrogen. For enzyme-substrate complexes, each solution was supplemented with HODPA (∼5-10 mm).Preliminary diffraction patterns were acquired with a typical laboratory instrument based on a rotating anode (Cu) generator equipped with focusing mirror or multilayer optics and an imaging plate detector (Rigaku/MSC). The diffraction data used for refinements of atomic models were acquired at SER-CAT beamline 22-ID-D at the Advanced Photon Source, Argonne National Laboratory. For the latter experiments, crystals were maintained at ∼100 K, and diffraction images were recorded by a MarMosaic 300 CCD detector (Mar USA, Inc., Evanston, IL). For each crystal, ∼100 frames were collected with a 1° rotation per frame; exposure times were 1-10 s per degree. All images were processed using DENZO, and then intensities were merged and scaled using SCALEPACK; both programs were from the HKL2000 program suite (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar).Structure Determination and Refinement—Programs from the CCP4 suite (26Bailey S. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar) were used for phasing and refinement. The crystal structure of wild-type BphD (PDB code 2OG1, Ref. 19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar, 24Seah S.Y.K. Terracina G. Bolin J.T. Riebel P. Snieckus V. Eltis L.D. J. Biol. Chem. 1998; 273: 22943-22949Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) served as a search model for phasing by molecular replacement using the program MOLREP (27Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4120) Google Scholar). Rigid body refinement was followed by iterative cycles of restrained atomic parameter refinement using REFMAC (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar) and manual density fitting using the molecular graphics program O (29Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). PRODRG (30Schuttelkopf A.W. van Aalten D.M.F. Acta Crystallogr. Sect. D: Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4142) Google Scholar) was used to develop structures of substrates and malonate for density fitting and establishment of refinement restraints. The deviation of restrained torsion angles from their expected values was used to evaluate the compatibility of the x-ray data with different tautomers of HOPDA. In the final refinement cycles of HOPDA complexes, the torsion angles in the non-aromatic portion of HOPDA were not restrained; bond lengths and bond angles were restrained to values expected for the keto (S112A: HOPDA) or enol (S112A/H265A:HOPDA) forms. The stereo-chemical properties of the models and the hydrogen bonding were analyzed by programs PROCHECK (31Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and REDUCE (32Word J.M. Lovell S.C. Richardson J.S. Richardson D.C. J. Mol. Biol. 1999; 285: 1735-1747Crossref PubMed Scopus (1078) Google Scholar).Optical Spectroscopy with Single Crystals—Visible light absorption spectroscopy was performed using crystals of the S112A and S112A/H265A variants exposed to HOPDA (∼15 mm in 30-60 μl of reservoir solution) for 30 min. The spectra were recorded at room temperature using a 4DX single crystal microspectrophotometer (4DX Systems) equipped with a MS125™ 1/8m spectrograph (Thermo Oriel), DB401 CCD detector (Andor Technologies), and a CLX 500 xenon lamp (Zeiss). Spectra were generated from the integration of one hundred 19-ms exposures recorded over the wavelength range 300-800 nm. Data acquisition and analysis was controlled by the Andor MCD software package supplied with the detector.After recording the spectra, the crystals were flash-frozen and diffraction data were measured at 100 K using the Rigaku/MSC equipment described above. The data were processed and electronic density maps were analyzed as described above.RESULTSKinetic Analysis of Variant Enzymes—Catalytic triad residues His-265 and Ser-112 were substituted to construct three variants: S112A, H265A, and S112A/H265A. The extremely low activity of the S112A and S112A/H265A variants prevented steady state kinetic measurements. Transformation of HOPDA by H265A as measured by the decay of absorbance at 434 nm could only be detected using large quantities of enzyme (∼1 μm), and the progress curve was biphasic. In an experiment in which 4 μm HOPDA was mixed with 1.3 μm H265A, the first phase could be described by a single exponential decay with a rate constant of 5.8 (±0.4) × 10-3 s-1. In Table 1 this is presented as the third rate of decay, or reciprocal relaxation time (1/τ3), because it is preceded by two events observed by stopped-flow spectrophotometry (see below). The value of 1/τ3 is similar to that of HOPDA tautomerization in solution, as observed by deuterium exchange NMR (19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar). The slope of the linear second phase approximately doubled upon increasing the H265A concentration to 2.6 μm. By contrast, doubling enzyme concentration did not affect the value of 1/τ3. The apparent burst is consistent with first order decay of an E:S complex followed by steady-state turnover. Curiously, the amplitude of the first phase corresponded to only 9 (±3)%of the total enzyme added, suggesting only this fraction of the active sites were functional. Similar behavior was observed in stopped-flow experiments (see below). Correcting for the active fraction of enzyme provides a rate of 0.0009 (±0.0002) s-1 for the steady state phase, which is about half of the kcat measured for the H265A variant of Ht-BphD (13Li J.J. Li C. Blindauer C.A. Bugg T.D.H. Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar). This is in reasonable agreement considering the current experiments were performed using a substrate concentration below the Km of 37 μm measured for Ht-BphD.TABLE 1Kinetic data for BphD variantsBphD variant1/τ11/τ21/τ3S112A/H265A220 s−1 (69%)22 s−1 (22%)0.34 s−1 (9%)H265A78 s−1 (82%)1.3 s−1 (18%)0.0058 s−1 (−100%)aMeasured using a Cary 5000 spectrophotometer as an exponential decay in absorbance at 434 nm.S112A~500 s−1 (−85%)76 s−1 (−11%)0.92 s−1 (−4%)a Measured using a Cary 5000 spectrophotometer as an exponential decay in absorbance at 434 nm. Open table in a new tab To better characterize the catalytic impairment of the variants, stopped-flow spectrophotometry was employed under single turnover conditions (E = 8 μm, S = 4 μm) at 25 °C. The S112A variant rapidly generated an intermediate with similar kinetics as the E:SRed intermediate transiently observed in wild type BphD (1/τ1 ∼ 500 s-1, Ref. 19Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (39) Google Scholar). The spectrum of E:SRed was more red-shifted and more intense in S112A (λmax = 506 nm; Table 1, Fig. 2A) than in the wild type (λmax = 492 nm, Fig. 2C). Moreover, E:SRed decayed extremely slowly in S112A, and was effectively trapped as an orange-colored complex. The half-life of this complex (S112A = 25 μm, HOPDA = 5 μm), determined by monitoring its absorbance at 506 nm, was 4.4 h at 25 °C.FIGURE 2Absorption spectra of intermediates in the BphD-catalyzed hydrolysis of HOPDA. A, spectra observed upon mixing 3.9 μm HOPDA (dashed line overlay) with 8 μm S112A. Spectra were recorded 1.1, 3.7, 8.8, 11, 14, 17, 19, 32, and 50 ms after mixing. The isosbestic point occurs at 461 nm. B, spectra observed upon mixing 3.4 μm HOPDA with 8 μm S112A/H265A. Spectra were recorded 3.7, 6.2, 8.8, 11, 14, and every ∼3 ms thereafter to 58 ms after mixing. C, spectral comparison of 4 μm HOPDA (solid line), the S112A:HOPDA complex (dashed line) and the E:SRed intermediate (dotted line) observed ∼20 ms after mixing 8 μm wild-type BphD with 4 μm HOPDA at 3.2 °C. Spectral intensities of the complexes were corrected based on Kd or rate constants. Spectra were recorded using potassium phosphate buffer (I = 0.1 m, pH 7.5) at 25" @default.
• W2019547185 created "2016-06-24" @default.
• W2019547185 creator A5002693857 @default.
• W2019547185 creator A5011192029 @default.
• W2019547185 creator A5028179990 @default.
• W2019547185 creator A5052838748 @default.
• W2019547185 creator A5063102320 @default.
• W2019547185 creator A5077634475 @default.
• W2019547185 date "2007-07-01" @default.
• W2019547185 modified "2023-09-27" @default.
• W2019547185 title "The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase" @default.
• W2019547185 cites W1539796472 @default.
• W2019547185 cites W1564644941 @default.
• W2019547185 cites W1839665474 @default.
• W2019547185 cites W1964441030 @default.
• W2019547185 cites W1979155909 @default.
• W2019547185 cites W1979984176 @default.
• W2019547185 cites W1986191025 @default.
• W2019547185 cites W1986332731 @default.
• W2019547185 cites W1987435485 @default.
• W2019547185 cites W1996410357 @default.
• W2019547185 cites W1996471792 @default.
• W2019547185 cites W2007232769 @default.
• W2019547185 cites W2007932555 @default.
• W2019547185 cites W2009285874 @default.
• W2019547185 cites W2013083986 @default.
• W2019547185 cites W2020551149 @default.
• W2019547185 cites W2024625551 @default.
• W2019547185 cites W2029207273 @default.
• W2019547185 cites W2037484441 @default.
• W2019547185 cites W2038840577 @default.
• W2019547185 cites W2039556766 @default.
• W2019547185 cites W2043941878 @default.
• W2019547185 cites W2055264790 @default.
• W2019547185 cites W2057422374 @default.
• W2019547185 cites W2062849327 @default.
• W2019547185 cites W2066486832 @default.
• W2019547185 cites W2080005671 @default.
• W2019547185 cites W2093363647 @default.
• W2019547185 cites W2097493124 @default.
• W2019547185 cites W2114367386 @default.
• W2019547185 cites W2115339329 @default.
• W2019547185 cites W2136327310 @default.
• W2019547185 cites W2145584804 @default.
• W2019547185 cites W2145732004 @default.
• W2019547185 cites W2159668414 @default.
• W2019547185 cites W2167551855 @default.
• W2019547185 cites W2414307244 @default.
• W2019547185 doi "https://doi.org/10.1074/jbc.m702237200" @default.
• W2019547185 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17442675" @default.
• W2019547185 hasPublicationYear "2007" @default.
• W2019547185 type Work @default.
• W2019547185 sameAs 2019547185 @default.
• W2019547185 citedByCount "34" @default.
• W2019547185 countsByYear W20195471852012 @default.
• W2019547185 countsByYear W20195471852013 @default.
• W2019547185 countsByYear W20195471852015 @default.
• W2019547185 countsByYear W20195471852017 @default.
• W2019547185 countsByYear W20195471852018 @default.
• W2019547185 countsByYear W20195471852019 @default.
• W2019547185 crossrefType "journal-article" @default.
• W2019547185 hasAuthorship W2019547185A5002693857 @default.
• W2019547185 hasAuthorship W2019547185A5011192029 @default.
• W2019547185 hasAuthorship W2019547185A5028179990 @default.
• W2019547185 hasAuthorship W2019547185A5052838748 @default.
• W2019547185 hasAuthorship W2019547185A5063102320 @default.
• W2019547185 hasAuthorship W2019547185A5077634475 @default.
• W2019547185 hasConcept C111233374 @default.
• W2019547185 hasConcept C181199279 @default.
• W2019547185 hasConcept C185592680 @default.
• W2019547185 hasConcept C2780120296 @default.
• W2019547185 hasConcept C55493867 @default.
• W2019547185 hasConcept C71240020 @default.
• W2019547185 hasConceptScore W2019547185C111233374 @default.
• W2019547185 hasConceptScore W2019547185C181199279 @default.
• W2019547185 hasConceptScore W2019547185C185592680 @default.
• W2019547185 hasConceptScore W2019547185C2780120296 @default.
• W2019547185 hasConceptScore W2019547185C55493867 @default.
• W2019547185 hasConceptScore W2019547185C71240020 @default.
• W2019547185 hasIssue "27" @default.
• W2019547185 hasLocation W20195471851 @default.
• W2019547185 hasOpenAccess W2019547185 @default.
• W2019547185 hasPrimaryLocation W20195471851 @default.
• W2019547185 hasRelatedWork W1973544677 @default.
• W2019547185 hasRelatedWork W2009120825 @default.
• W2019547185 hasRelatedWork W2012958978 @default.
• W2019547185 hasRelatedWork W2056511259 @default.
• W2019547185 hasRelatedWork W2064425605 @default.
• W2019547185 hasRelatedWork W2318628289 @default.
• W2019547185 hasRelatedWork W2397827493 @default.
• W2019547185 hasRelatedWork W2949507347 @default.
• W2019547185 hasRelatedWork W2950570661 @default.
• W2019547185 hasRelatedWork W2952037489 @default.
• W2019547185 hasVolume "282" @default.
• W2019547185 isParatext "false" @default.
• W2019547185 isRetracted "false" @default.
• W2019547185 magId "2019547185" @default.
• W2019547185 workType "article" @default.