FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2062387625> ?p ?o ?g. }

• W2062387625 endingPage "36385" @default.
• W2062387625 startingPage "36377" @default.
• W2062387625 abstract "The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A·3-Cl HOPDA and S112A·3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A·HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 Å from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A·3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation. The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A·3-Cl HOPDA and S112A·3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A·HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 Å from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A·3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation. Polychlorinated biphenyls (PCBs) 4The abbreviations used are:PCBpolychlorinated biphenylBphbiphenylDHBdihydroxybiphenylDHBDDHB dioxygenaseHOPDA2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acidWTwild type. 4The abbreviations used are:PCBpolychlorinated biphenylBphbiphenylDHBdihydroxybiphenylDHBDDHB dioxygenaseHOPDA2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acidWTwild type. were manufactured extensively in the 20th century for industrial and commercial applications, including use in electrical transformers, hydraulics, and plasticizers. 5U. S. Environmental Protection Agency (Oct. 24, 2007) Polychlorinated biphenyls (PCBs) (Oct. 24, 2007) http://www.epa.gov/epaoswer/hazwaste/pcbs/index.htm. 5U. S. Environmental Protection Agency (Oct. 24, 2007) Polychlorinated biphenyls (PCBs) (Oct. 24, 2007) http://www.epa.gov/epaoswer/hazwaste/pcbs/index.htm. Although banned in the United States since 1977, environmentally persistent PCBs have been linked to cancer (2Knerr S. Schrenk D. Crit. Rev. Toxicol. 2006; 36: 663-694Crossref PubMed Scopus (122) Google Scholar), childhood neurodevelopmental deficits arising from prenatal exposure (3Jacobson J.L. Jacobson S.W. N. Engl. J. Med. 1996; 335: 783-789Crossref PubMed Scopus (970) Google Scholar), and a host of other effects attributed to endocrine disruption (4Toft G. Hagmar L. Giwercman A. Bonde J.P. Reprod. Toxicol. 2004; 19: 5-26Crossref PubMed Scopus (140) Google Scholar). Indeed, concerns about high concentrations of PCBs and other contaminants in some freshwater fish have prompted a recent comprehensive risk-benefit analysis of fish consumption (5Mozaffarian D. Rimm E.B. J. Am. Med. Assoc. 2006; 296: 1885-1899Crossref PubMed Scopus (1524) Google Scholar).The observation that bacteria partially degrade PCBs has motivated research into microbial bioremediation as an alternative to traditional remediation approaches, which typically require the costly and invasive removal of contaminated soil. Bacteria use the biphenyl (Bph) pathway to aerobically degrade many PCBs in a strain-dependent manner. The upper Bph pathway consists of four enzymes that transform biphenyl into benzoic acid and 2-hydroxypenta-2,4-dienoic acid (6Pieper D.H. Appl. Microbiol. Biotechnol. 2005; 67: 170-191Crossref PubMed Scopus (283) Google Scholar). Nevertheless, certain congeners are not efficiently degraded by this pathway, leading to the accumulation of these congeners or their chlorinated metabolites and even inhibition of Bph enzymes. For example, 2′,6′-diCl 2,3-dihydroxybiphenyl (2′,6′-diCl-DHB) inhibits the third pathway enzyme, DHB dioxygenase (DHBD) in the potent PCB-degrading strain Burkholderia xenovorans LB400 (7Dai S.D. Vaillancourt F.H. Maaroufi H. Drouin H.M. Neau D.B. Snieckus V. Bolin J.T. Eltis L.D. Nat. Struct. Biol. 2002; 9: 934-939Crossref PubMed Scopus (80) Google Scholar). Similarly, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids (HOPDAs) that are chlorinated at the 3 and 4 positions inhibit the fourth enzyme, BphD, of B. xenovorans LB400 (8Seah 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 (83) Google Scholar) and Rhodococcus globerulus P6 (8Seah 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 (83) Google Scholar, 9Seah S.Y.K. Ke J. Denis G. Horsman G.P. Fortin P.D. Whiting C.J. Eltis L.D. J. Bacteriol. 2007; 189: 4038-4045Crossref PubMed Scopus (29) Google Scholar, 10Seah S.Y.K. Labbé G. Kaschabek S.R. Reifenrath F. Reineke W. Eltis L.D. J. Bacteriol. 2001; 183: 1511-1516Crossref PubMed Scopus (45) Google Scholar) as described below. Interestingly, a glutathione S-transferase in B. xenovorans LB400, BphK, catalyzes the dehalogenation of 3-Cl HOPDA (11Fortin P.D. Horsman G.P. Yang H.M. Eltis L.D. J. Bacteriol. 2006; 188: 4424-4430Crossref PubMed Scopus (24) Google Scholar) and DxnB2, a homolog of BphD from the dibenzofuran catabolic pathway of Sphingomonas wittichii RW1, catalyzes the hydrolysis of 3-Cl HOPDA (8Seah 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 (83) Google Scholar, 9Seah S.Y.K. Ke J. Denis G. Horsman G.P. Fortin P.D. Whiting C.J. Eltis L.D. J. Bacteriol. 2007; 189: 4038-4045Crossref PubMed Scopus (29) Google Scholar, 10Seah S.Y.K. Labbé G. Kaschabek S.R. Reifenrath F. Reineke W. Eltis L.D. J. Bacteriol. 2001; 183: 1511-1516Crossref PubMed Scopus (45) Google Scholar). Nevertheless, chlorinated HOPDAs accumulated in whole cell studies of PCB degradation (12Furukawa K. Tomizuka N. Kamibayashi A. Appl. Environ. Microb. 1979; 38: 301-310Crossref PubMed Google Scholar, 13Furukawa K. Tonomura K. Kamibayashi A. Appl. Environ. Microb. 1978; 35: 223-227Crossref PubMed Google Scholar, 14Seeger M. Timmis K.N. Hofer B. Appl. Environ. Microb. 1995; 61: 2654-2658Crossref PubMed Google Scholar), suggesting that BphD represents a bottle-neck for PCB degradation.The inhibition of Bph enzymes has recently taken on additional significance in light of discoveries in Mycobacterium tuberculosis, the etiological agent of tuberculosis. Specifically, homologues of the Bph enzymes are involved in cholesterol catabolism (15Van 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 (405) Google Scholar) and are critical for the survival of the pathogen in the human macrophage (16Rengarajan J. Bloom B.R. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8327-8332Crossref PubMed Scopus (551) Google Scholar). The lack of human homologues of the cholesterol-degrading enzymes suggests that they are promising drug targets. Understanding the mechanism of BphD inhibition will facilitate the engineering of the enzyme to improve its activity toward chlorinated substrates and should inform the development of inhibitors of HsaD, the cholesterol-degrading homologue in M. tuberculosis.BphD catalyzes the hydrolytic C-C bond cleavage of HOPDA (Fig. 1) and has features typical of the α/β-hydrolase superfamily (17Holmquist M. Curr. Prot. Pept. Sci. 2000; 1: 209-235Crossref PubMed Scopus (472) Google Scholar, 18Ollis 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), including the fold and conserved active site catalytic triad composed of serine, histidine, and aspartate residues. To expel the electron-rich dienoate moiety of the substrate, the enzyme first employs a His-265-mediated enol-keto tautomerization to generate a hydrolyzable keto-intermediate (E·Sk) (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (38) Google Scholar, 21Henderson I.M.J. Bugg T.D.H. Biochemistry. 1997; 36: 12252-12258Crossref PubMed Scopus (38) Google Scholar, 22Lam W.W.Y. Bugg T.D.H. Biochemistry. 1997; 36: 12242-12251Crossref PubMed Scopus (48) Google Scholar, 23Li 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, 24Li J.J. Li C. Blindauer C.A. Bugg T.D.H. Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar). Indeed, His-265-dependent formation of an intermediate with a red-shifted absorbance spectrum (E·Sred) is formed at a similar rate in both WT and S112A BphDs (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Crystallographic data for the E·Sred intermediate trapped in the S112A·HOPDA complex are most consistent with the bound HOPDA being ketonized (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), although a non-planar, distorted conformation of the enol/enolate could not be completely ruled out. Hydrolysis then proceeds via either a gem-diol intermediate (24Li J.J. Li C. Blindauer C.A. Bugg T.D.H. Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar, 25Fleming S.M. Robertson T.A. Langley G.J. Bugg T.D.H. Biochemistry. 2000; 39: 1522-1531Crossref PubMed Scopus (55) Google Scholar, 26Li J.J. Bugg T.D.H. Org. Biomol. Chem. 2007; 5: 507-513Crossref PubMed Google Scholar, 27Speare 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, 28Speare D.M. Olf P. Bugg T.D.H. Chem. Commun. 2002; : 2304-2305Crossref PubMed Google Scholar) or an acyl-enzyme intermediate (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (38) Google Scholar). As noted above, HOPDAs that are chlorinated on the dienoate moiety are poorly transformed by BphD (8Seah 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 (83) Google Scholar, 9Seah S.Y.K. Ke J. Denis G. Horsman G.P. Fortin P.D. Whiting C.J. Eltis L.D. J. Bacteriol. 2007; 189: 4038-4045Crossref PubMed Scopus (29) Google Scholar, 10Seah S.Y.K. Labbé G. Kaschabek S.R. Reifenrath F. Reineke W. Eltis L.D. J. Bacteriol. 2001; 183: 1511-1516Crossref PubMed Scopus (45) Google Scholar). Specifically, 5-chlorination reduced the maximal rate of BphD by 3-fold, and chlorination at the 3 or 4 positions reduced the maximal rate by ∼103 and ∼104-fold, respectively. Although 4-Cl HOPDA is the least efficiently transformed monochlorinated HOPDA, 3-Cl HOPDAs represent a more significant roadblock to PCB degradation in two respects. First, 3-Cl HOPDA (t½ ∼ 500 h) is more stable than 4-Cl HOPDA, which undergoes a non-enzymatic transformation to 4-OH HOPDA (t½ = 2.8 h) followed by degradation to products that include acetophenone (t½ ∼ 180 h) (8Seah 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 (83) Google Scholar). Second, 3-Cl HOPDA is a very poor substrate of BphDLB400, effectively inhibiting the hydrolysis of HOPDA (Kic = 0.57 μm) more potently than either 4-Cl HOPDA (Kic = 3.6 μm) or 4-OH HOPDA (Kic = 0.95 μm). It has been unclear whether the 3-Cl substituent impairs tautomerization or hydrolysis and whether the defect is due to an electronic or steric effect.Herein we investigated the basis for inhibition of BphD by 3-Cl HOPDA. First, the ability of BphD to hydrolyze a series of 3-substituted HOPDAs was studied using steady-state kinetics to compare steric versus electronic effects. Second, single turnover stopped-flow kinetic analysis was used to probe the effects of 3-substitution on E·Sred formation in both WT and hydrolytically impaired Ser112Ala variants. Finally, crystal structures of the S112A·3-Cl HOPDA and S112A·3,10-diF-HOPDA complexes were analyzed to reveal the structural correlates of the kinetic behavior.MATERIALS AND METHODSChemicals—HOPDA, 3-Cl HOPDA, 3-Me HOPDA, and 3,10-diF HOPDA were enzymatically generated from DHB, 4-Cl DHB, 4-Me DHB, and 4,4′-diF DHB, respectively, using DHBD as previously described (8Seah 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 (83) Google Scholar, 20Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (38) Google Scholar). The preparation of DHB and chlorinated DHBs has been described elsewhere (29Nerdinger 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). All other chemicals were of analytical grade.Preparation of 4,4′-diF DHB—A plate of W medium (30Seto M. Kimbara K. Shimura M. Hatta T. Fukuda M. Yano K. Appl. Environ. Microb. 1995; 61: 3353-3358Crossref PubMed Google Scholar) was streaked with frozen stock of Pandoraea pnomenusa (formerly Comamonas testosteroni) B-356 and incubated with biphenyl crystals in the lid at 30 °C until colonies were visible (∼5 days). Several colonies were added to 3 ml of W medium containing 1 mg biphenyl and incubated at 30 °C and 250 rpm until cloudy (∼4 days). Alternatively, this step could be shortened to ∼2 days if a 50-μl aliquot of frozen stock was used to inoculate the 3 ml of culture. One liter of W medium containing 0.5 g of biphenyl in a 2-liter flask was inoculated with 1 ml of starter culture, and the mixture was incubated as above. When the optical density at 600 nm reached 1, the culture was carefully decanted to remove biphenyl crystals and then centrifuged for 10 min at 7000 × g. The pellet was washed two times with potassium phosphate buffer (I = 0.1 m, pH 7.5) to remove residual biphenyl and resuspended in 500 ml of buffer supplemented with 100 mg of 4,4′-diF-biphenyl and 7 mg of 3-chlorocatechol. The latter was included to inactivate DHBD (31Vaillancourt F.H. Labbe G. Drouin N.M. Fortin P.D. Eltis L.D. J. Biol. Chem. 2002; 277: 2019-2027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and thereby prevent enzymatic transformation of the produced 4,4′-diF-DHB. The culture was incubated as above, and 50-μl aliquots were analyzed by high-performance liquid chromatography at 30-min intervals using a Prodigy ODS Prep column, 2.1 × 250 mm (Phenomenex, Torrance, CA), operating at a flow rate of 1.5 ml/min. The mobile phase initially consisted of a 30:70 ratio of solvent A (0.5% aqueous H3PO4) to solvent B (methanol) for the first 5 min of the run, then a gradient was used to achieve 100% B at 10 min. The retention time of 3-chlorocatechol was 2.6 min, 4,4′-diF-DHB eluted at 5.4 min, and 4,4′-diF-biphenyl eluted at 13 min. When the maximum concentration of 4,4′-diF-DHB in the culture was reached as judged by high-performance liquid chromatography (after ∼3 h), the culture was filtered to remove undissolved starting material, and then extracted three times with ∼200 ml of ethyl acetate. The pooled fractions were dried over anhydrous MgSO4 and rotary evaporated to dryness. The crude extract was dissolved in an appropriate volume of mobile phase (20:80 A:B ratio) and purified by preparative high-performance liquid chromatography using a Prodigy ODS Prep column, 21.2 × 250 mm (Phenomenex, Torrance, CA) operating at a flow rate of 8.5 ml/min. The 4,4′-diF-DHB (retention time ∼ 8 min) was collected, extracted into ethyl acetate, dried, and evaporated as above. The purity was estimated to be >95% by high-performance liquid chromatography. The 4,4′-diF-DHB was dissolved in 10% ethanol, 80% H2O, and 10% D2O to collect NMR spectra at 25 °C using a 600-MHz spectrometer at the Department of Chemistry, University of Rochester. The 1H NMR reference compound was 100 μm 2,2-dimethyl-2-silapentane 5-sulfonate and was used as an indirect reference for 19F NMR (found by 19F{1H} NMR: -3.6 (s, 1F), -23.9 (s, 1F)) and 1H NMR (6.75-6.82 (m, 2H), 7.21 (t, 2H, J = 6 Hz), 7.52 (t, 2H, J = 6 Hz)).Steady-state Kinetic Measurements—Initial rates of BphD-catalyzed hydrolysis at varying substrate concentrations were obtained by monitoring the substrate absorbance maximum versus time in potassium phosphate buffer (I = 0.1 m, pH 7.5) using a Varian Cary 5000 spectrophotometer equipped with a thermostatted cuvette holder (Varian Canada, Mississauga, Ontario, Canada) maintained at 25.0 ± 0.5 °C, controlled by Cary WinUV software version 2.00. Reactions were carried out in a 1-ml volume and were initiated by the addition of 5 μl of an appropriately diluted enzyme solution. The 3-Me HOPDA was generated in situ by adding 80 μg of polyhistidine-tagged DHBD to the cuvette containing 4-Me DHB. After obtaining the rate of background decay (0.5-1 min), BphD was added and the activity of the enzyme was determined by correcting for the background. The Michaelis-Menten equation was fit to the data using LEONORA (32Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, New York1995Google Scholar). The molar absorptivity of 3,10-diF HOPDA (ϵ438 = 37.9 ± 2.1 mm-1 cm-1) was calculated from its absorption spectrum after quantification by measuring the amount of dioxygen consumed in the ring-opening reaction of 4,4′-diF DHB catalyzed by DHBD. Dioxygen consumption was measured using a Clark-type polarographic oxygen electrode (Yellow Springs Instruments model 5301, Yellow Springs, OH) as previously described for other HOPDAs (33Seah 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 (67) Google Scholar). The molar absorptivity of 3-Me HOPDA (ϵ430 = 18.7 ± 0.4 mm-1 cm-1) was determined by recording the absorption spectrum of a known quantity of 4-Me DHB immediately after cleavage by DHBD.Stopped-flow Spectrophotometry—Single turnover reactions (BphD = 8 μm; substrate = 4 μm) in the phosphate buffer described above were monitored 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 recirculating water system. Multiple wavelength data from the time courses of single shots were acquired using the Xscan software (Applied Photophysics Ltd.), then saved as CSV files in the RISC Pro-K software and exported to Excel where replicate measurements from at least four shots were averaged. Selected single wavelength datasets were then imported into the SX18MV software (Applied Photophysics Ltd.) where multiple exponential equations were fit to the data to obtain reciprocal relaxation times and amplitudes. Good fits were characterized by random variation in the residuals.Crystallization and Preparation of Complexes—Crystals of the substrate-free S112A variant of BphD were grown at 20 °C in 1.9 m sodium malonate, pH 7.0, by sitting drop vapor diffusion, as previously reported (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Complexes were prepared by incubating crystals for 30 min in 60 μl of reservoir solution supplemented with ∼10 mm 3-Cl-HOPDA or ∼65 mm 3,10-difluoro-HOPDA. Crystals were prepared for flash freezing by serial transfer into solutions containing higher concentrations of sodium malonate (3.4 m and 3.7 m, pH 7.0) augmented with ∼5 mm of 3-Cl HOPDA or trace amounts of 3,10-difluoro-HOPDA. The incubation time was 3-6 s per step. Crystals were frozen by immersion in liquid N2.Diffraction Experiments and Structure Analysis—All diffraction data were acquired by the use of the SERCAT facilities at the Advanced Photon Source, Argonne National Laboratory. The x-ray wavelength was 1.0 Å, and crystals were maintained at ∼100 K during data collection. Diffraction images were recorded by a MarMosaic 300 charge-coupled device 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 intensities were merged and scaled using SCALEPACK; both programs were from the HKL2000 program suite (34Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar).The initial model for both complexes included only the protein atoms from the crystal structure of the S112A·malonate complex (PDB code 2PU6) (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Rigid body refinement performed in REFMAC (35Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar) from the CCP4 package (36Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar) was followed by iterative cycles of restrained atomic parameter refinement via REFMAC and manual density fitting using the molecular graphics program O (37Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). PRODRG (38Schuttelkopf A.W. van Aalten D.M.F. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4136) Google Scholar) was used to construct structures of 3-Cl HOPDA, 3,10-difluoro-HODPA, and malonate for density fitting and establishment of refinement restraints. The bond lengths and bond angles of the ligands were restrained to values expected for the enol isomer. Torsion angles in the non-aromatic portion of the substrates were unrestrained. The stereochemical properties of the models and the hydrogen bonding were analyzed by using the programs PROCHECK (39Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and REDUCE (40Word J.M. Lovell S.C. Richardson J.S. Richardson D.C. J. Mol. Biol. 1999; 285: 1735-1747Crossref PubMed Scopus (1078) Google Scholar).RESULTSSteady-state Kinetics—To assess the basis of inhibition of BphD by 3-Cl HOPDA, the enzyme-catalyzed hydrolysis of 3,10-diF HOPDA and 3-Me HOPDA was studied using steady-state kinetics. The 10-fluoro substituent is not expected to greatly affect catalysis because HOPDAs with small, electron-withdrawing substituents (e.g. -Cl and -CF3) at this position are hydrolyzed by BphD with kcat values within 25% of that for HOPDA (8Seah 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 (83) Google Scholar, 28Speare D.M. Olf P. Bugg T.D.H. Chem. Commun. 2002; : 2304-2305Crossref PubMed Google Scholar). Significantly, reduction of the volume of the electronegative 3-substituent by 40% upon chlorine-to-fluorine substitution resulted in a 150-fold increase in kcat (Table 1), indicating that larger 3-substituents interfere with catalysis. This conclusion was supported by the observation that the substrate with the largest 3-substituent, 3-Me HOPDA, had the lowest apparent kcat (Table 1). Although a Km value was reported for 3-Cl HOPDA (8Seah 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 (83) Google Scholar), Km could not be determined for 3-Me HOPDA due to a combination of lower activity, lower molar absorptivity, and higher background decay rates, which together prevented reliable initial rate measurements at low substrate concentrations. Finally, we also determined the stability of HOPDAs in the enzyme reaction buffer (Table 1), but we discerned no obvious correlation between either the electronegativity of the substituents and the nonenzymatic decomposition, or between the nonenzymatic and enzymatic reactions. In conclusion, kcat was strongly anticorrelated with the volume of the substituent at C3 and uncorrelated with electronegativity over the series of HOPDAs studied, suggesting that turnover is largely dictated by steric, not electronic, factors.TABLE 1Steady-state parameters of BphD hydrolysis of 3-substituted HOPDAs Errors are less than 15%.3-X HOPDAHalf-lifeElectronegativityaFrom Ref. 43VolumebFrom Ref. 44C-X bond lengthcFrom Ref. 45kcatKmkcat/KmXhÅ3Ås-1μmμm-1 s-1H58dFrom Ref. 82.27.21.086.50.232F114.113.31.341.44.80.29Cl500dFrom Ref. 82.822.51.730.00890.540.016Me302.3eFrom Ref. 4628.41.510.0036fEstimated from initial rate measurements at high substrate concentrations ([S] » Km)NDgND, not determinedNDa From Ref. 43Allred A.L. Rochow E.G. J. Inorg. Nucl. Chem. 1958; 5: 215-221Google Scholarb From Ref. 44Zhao Y.H. Abraham M.H. Zissimos A.M. J. Org. Chem. 2003; 68: 7368-7373Crossref PubMed Scopus (462) Google Scholarc From Ref. 45Allen F.H. Kennard O. Watson D.G. Brammer L. Orpen A.G. Taylor R. J. Chem. Soc. Perkin Trans. 1987; 2: S1-S19Crossref Google Scholard From Ref. 8Seah 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 (83) Google Scholare From Ref. 46Liu H. Wang Q.W. Liu L.X. J. Chem. Educ. 1992; 69: 783-784Crossref Google Scholarf Estimated from initial rate measurements at high substrate concentrations ([S] » Km)g ND, not determined Open table in a new tab Stopped-flow Kinetics—To better elucidate the influence of 3-substituents on catalysis, stopped-flow experiments were conducted to resolve individual catalytic steps. In a previous single turnover ([E] > [S]) stopped-flow experiment using WT BphD and HOPDA, we observed rapid (∼500 s-1) formation of E·Sred (λmax = 492 nm) from the free HOPDA enolate (λmax = 434 nm) (Fig. 2E) (20Horsman G.P. Ke J. Dai S. Seah S.Y.K. Bolin J.T. Eltis L.D. Biochemistry. 2006; 45: 11071-11086Crossref PubMed Scopus (38) Google Scholar). A similar E·Sred intermediate (λmax = 506 nm) was rapidly formed (∼500 s-1) and trapped in the hydrolytically impaired S112A variant (Fig. 2F), and crystallographically was observed in a non-planar conformation (19Horsman G.P. Bhowmik S. Seah S.Y.K. Kumar P. Bolin J.T. Eltis L.D. J. Biol. Chem. 2007; 282: 19894-19904Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In contrast, E·Sred did not accumulate when 3-Cl HOPDA was used as a substrate (Fig. 2A). Under single turnover conditions ([BphD] = 8 μm, [3-Cl HOPDA] = 4 μm), an initial decrease (1/τ1 = 15 s-1) occurred at the absorbance maximum of the 3-Cl HOPDA enolate (432 nm) (Table 2). This decay corresponded to a ∼20% loss in absorbance and occurred together with a slight blue shift to 427 nm. This may correspond t" @default.
• W2062387625 created "2016-06-24" @default.
• W2062387625 creator A5002693857 @default.
• W2062387625 creator A5028179990 @default.
• W2062387625 creator A5063102320 @default.
• W2062387625 creator A5077634475 @default.
• W2062387625 date "2007-12-01" @default.
• W2062387625 modified "2023-10-11" @default.
• W2062387625 title "The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation" @default.
• W2062387625 cites W1539796472 @default.
• W2062387625 cites W1571304687 @default.
• W2062387625 cites W1578099536 @default.
• W2062387625 cites W1839665474 @default.
• W2062387625 cites W1969178306 @default.
• W2062387625 cites W1977160749 @default.
• W2062387625 cites W1979155909 @default.
• W2062387625 cites W1979984176 @default.
• W2062387625 cites W1981418080 @default.
• W2062387625 cites W1986191025 @default.
• W2062387625 cites W1986332731 @default.
• W2062387625 cites W1987435485 @default.
• W2062387625 cites W2007932555 @default.
• W2062387625 cites W2013083986 @default.
• W2062387625 cites W2017848267 @default.
• W2062387625 cites W2019086744 @default.
• W2062387625 cites W2019547185 @default.
• W2062387625 cites W2020429677 @default.
• W2062387625 cites W2024625551 @default.
• W2062387625 cites W2033642038 @default.
• W2062387625 cites W2037484441 @default.
• W2062387625 cites W2038840577 @default.
• W2062387625 cites W2039556766 @default.
• W2062387625 cites W2043941878 @default.
• W2062387625 cites W2055264790 @default.
• W2062387625 cites W2057422374 @default.
• W2062387625 cites W2062849327 @default.
• W2062387625 cites W2066486832 @default.
• W2062387625 cites W2069929526 @default.
• W2062387625 cites W2093363647 @default.
• W2062387625 cites W2095064953 @default.
• W2062387625 cites W2104049876 @default.
• W2062387625 cites W2109231516 @default.
• W2062387625 cites W2113325056 @default.
• W2062387625 cites W2114367386 @default.
• W2062387625 cites W2115339329 @default.
• W2062387625 cites W2135150658 @default.
• W2062387625 cites W2148180351 @default.
• W2062387625 cites W2151307812 @default.
• W2062387625 cites W2155155225 @default.
• W2062387625 cites W2159668414 @default.
• W2062387625 cites W2171093181 @default.
• W2062387625 cites W2330340734 @default.
• W2062387625 cites W2414307244 @default.
• W2062387625 doi "https://doi.org/10.1074/jbc.m707035200" @default.
• W2062387625 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17932031" @default.
• W2062387625 hasPublicationYear "2007" @default.
• W2062387625 type Work @default.
• W2062387625 sameAs 2062387625 @default.
• W2062387625 citedByCount "22" @default.
• W2062387625 countsByYear W20623876252012 @default.
• W2062387625 countsByYear W20623876252013 @default.
• W2062387625 countsByYear W20623876252015 @default.
• W2062387625 countsByYear W20623876252016 @default.
• W2062387625 countsByYear W20623876252017 @default.
• W2062387625 countsByYear W20623876252018 @default.
• W2062387625 countsByYear W20623876252019 @default.
• W2062387625 countsByYear W20623876252020 @default.
• W2062387625 crossrefType "journal-article" @default.
• W2062387625 hasAuthorship W2062387625A5002693857 @default.
• W2062387625 hasAuthorship W2062387625A5028179990 @default.
• W2062387625 hasAuthorship W2062387625A5063102320 @default.
• W2062387625 hasAuthorship W2062387625A5077634475 @default.
• W2062387625 hasBestOaLocation W20623876251 @default.
• W2062387625 hasConcept C181199279 @default.
• W2062387625 hasConcept C185592680 @default.
• W2062387625 hasConcept C2779679103 @default.
• W2062387625 hasConcept C2780120296 @default.
• W2062387625 hasConcept C41008148 @default.
• W2062387625 hasConcept C55493867 @default.
• W2062387625 hasConcept C71240020 @default.
• W2062387625 hasConcept C76155785 @default.
• W2062387625 hasConceptScore W2062387625C181199279 @default.
• W2062387625 hasConceptScore W2062387625C185592680 @default.
• W2062387625 hasConceptScore W2062387625C2779679103 @default.
• W2062387625 hasConceptScore W2062387625C2780120296 @default.
• W2062387625 hasConceptScore W2062387625C41008148 @default.
• W2062387625 hasConceptScore W2062387625C55493867 @default.
• W2062387625 hasConceptScore W2062387625C71240020 @default.
• W2062387625 hasConceptScore W2062387625C76155785 @default.
• W2062387625 hasIssue "50" @default.
• W2062387625 hasLocation W20623876251 @default.
• W2062387625 hasOpenAccess W2062387625 @default.
• W2062387625 hasPrimaryLocation W20623876251 @default.
• W2062387625 hasRelatedWork W1840663199 @default.
• W2062387625 hasRelatedWork W1964441030 @default.
• W2062387625 hasRelatedWork W1970974546 @default.
• W2062387625 hasRelatedWork W1995816032 @default.
• W2062387625 hasRelatedWork W1996140555 @default.

• next