Purification and Preliminary Characterization of a Serine Hydrolase Involved in the Microbial Degradation of Polychlorinated Biphenyls

Seah, Stephen Y. K.; Terracina, Giuseppe; Bolin, Jeffrey T.; Riebel, Peter; Snieckus, Victor; Eltis, Lindsay D.

doi:10.1074/jbc.273.36.22943

Cited by 70 publications

(95 citation statements)

References 43 publications

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“…Quaternary structure-As observed in solution (24), BphD LB400 is a tetrameric oligomer in the crystal ( Figure 6). The tetramer is formed by the back-to-back interaction of two A-B dimers related by crystallographic two-fold symmetry.…”

Section: Crystal Structuresmentioning

confidence: 63%

“…All other chemicals were of analytical grade. Catechol 2,3-dioxygenase was used to prepare 2-hydroxy-6-oxo-2,4-heptadienoic acid from 3-methylcatechol as previously described (23).…”

Section: Chemicalsmentioning

confidence: 99%

“…The active site serine of BphD was mutated to cysteine (S112C) using the Transformer site-directed mutagenesis method (Clontech Laboratories, Palo Alto, CA, USA). Briefly, the S112C mutagenic primer (primer S112C: 5'-GCGCCCCCCATGCAGTTGCCGACCAG-3') and a second mutagenic selection primer to remove an EcoRI site (primer ODM: 5'-AGCTCGAATTGGTAATCATGG-3') were mixed with the pEMBL18 plasmid containing the BphD LB400 gene (pSS184) (23). After second strand synthesis and ligation using T4 DNA polymerase and T4 DNA ligase (Pharmacia, Uppsala, Sweden), respectively, the DNA was digested with EcoRI to linearize unmutated plasmid, and transformed into E. coli BMH 71−18 mutS.…”

Section: Mutagenesis Protein Expression and Purificationmentioning

confidence: 99%

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Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

et al. 2006

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Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD LB400 ) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 °C showed that the enzyme rapidly (1/τ 1 ∼ 500 s −1 ) transforms HOPDA (λ max = 434 nm) to a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/τ 2 = 54 s −1 , 1/τ 3 = 6 s −1 ∼ k cat ) with simultaneous biphasic appearance (48 and 8 s −1 ) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/τ 1 and 1/τ 2 , but affected neither 1/τ 3 nor k cat , suggesting that 1/τ 2 may reflect diffusive HPD dissociation, while 1/τ 3 represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and therefore postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (λ max = 492 nm). Crystal structures of wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 Å resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogenbonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.The microbial degradation of aromatic compounds is crucial to maintaining the global carbon cycle (1). Aerobic degradation typically involves oxygenation of the aromatic ring to produce a catechol followed by a dioxygenase-catalyzed ring-opening reaction. In one type of degradation pathway, ring-opening yields a meta-cleavage product (MCP) 1 . A serine hydrolase † This work was funded by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant) and the National Institutes of Health (GM-52381).*To whom correspondence should be addressed: Lindsay D. Eltis, leltis@interchange.ubc.ca, Phone: (604) Fax: (604)822 −6041. § These authors contributed equally to this work. ¶ Present addresses: JK: University of Kentucky, Lexington, KY 40506, USA; SD: Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, CO 80206; SS: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada 1 Abbreviations: MCP, meta-cleavage product; HPD, 2-hydroxy-penta-2,4-dienoic acid; PCB, polychlorinated biphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid; DHB, 2,3-dihydroxybiphenyl; DHBD, dihydroxybiphenyl dioxygenase; S k , ketonized substrate; E:S k , enzyme-bound ketonized substrate; P...

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Section: Crystal Structuresmentioning

confidence: 63%

“…All other chemicals were of analytical grade. Catechol 2,3-dioxygenase was used to prepare 2-hydroxy-6-oxo-2,4-heptadienoic acid from 3-methylcatechol as previously described (23).…”

Section: Chemicalsmentioning

confidence: 99%

Section: Mutagenesis Protein Expression and Purificationmentioning

confidence: 99%

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Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

et al. 2006

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“…It has been proposed that the keto form of the substrate binds weakly and can dissociate from the active site prior to attack by water. Steady-state kinetic studies on BphD of B. cepacia LB400 (Bph-D LB400 ) indicate that this enzyme is also "leaky" (24).…”

Section: Pcbsmentioning

confidence: 99%

Identification of a Serine Hydrolase as a Key Determinant in the Microbial Degradation of Polychlorinated Biphenyls

Seah¹,

Labbé²,

Nerdinger³

et al. 2000

Journal of Biological Chemistry

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100

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The ability of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) of Burkholderia cepacia LB400 to hydrolyze polychlorinated biphenyl (PCB) metabolites was assessed by determining its specificity for monochlorinated HOPDAs. The relative specificities of BphD for HOPDAs bearing chlorine substituents on the phenyl moiety were 0.28, 0.38, and 1.1 for 8-Cl, 9-Cl, and 10-Cl HOPDA, respectively, versus HOPDA (100 mM phosphate, pH 7.5, 25°C). In contrast, HOPDAs bearing chlorine substituents on the dienoate moiety were poor substrates for BphD, which hydro-

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“…The molar absorptivity of 3,10-diF HOPDA (⑀ 438 ϭ 37.9 Ϯ 2.1 mM Ϫ1 cm Ϫ1 ) was calculated from its absorption spectrum after quantification by measuring the amount of dioxygen consumed in the ring-opening reaction of 4,4Ј-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 (33). The molar absorptivity of 3-Me HOPDA (⑀ 430 ϭ 18.7 Ϯ 0.4 mM Ϫ1 cm Ϫ1 ) was determined by recording the absorption spectrum of a known quantity of 4-Me DHB immediately after cleavage by DHBD.…”

Section: Methodsmentioning

confidence: 99%

The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation

Bhowmik

Horsman

Bolin

et al. 2007

Journal of Biological Chemistry

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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 S112A⅐3-Cl HOPDA and S112A⅐3,10-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 S112A⅐HOPDA 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 S112A⅐3-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.

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Purification and Preliminary Characterization of a Serine Hydrolase Involved in the Microbial Degradation of Polychlorinated Biphenyls

Cited by 70 publications

References 43 publications

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

Identification of a Serine Hydrolase as a Key Determinant in the Microbial Degradation of Polychlorinated Biphenyls

The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation

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