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

Seah, Stephen Y. K.; Labbé, Geneviève; Nerdinger, Sven; Johnson, Matthew R.; Snieckus, Victor; Eltis, Lindsay D.

doi:10.1074/jbc.275.21.15701

Cited by 93 publications

(109 citation statements)

References 33 publications

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“…By contrast, almost complete deuterium incorporation occurred at H-5 E , as predicted. 3 Hence the enzyme stereospecifically incorporates a proton at H-5 E , and the non-specific incorporation at H-5 Z can be explained by non-enzymatic exchange prior to enzymatic hydrolysis. If the reaction was allowed to continue after the complete conversion of HOPDA, ketonized HPD ((E)-2-oxo-3-pentenoate) appeared in the NMR spectrum (data not shown).…”

Section: Deuterium Incorporation Into Hpdmentioning

confidence: 99%

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: Deuterium Incorporation Into Hpdmentioning

confidence: 99%

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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“…Chemicals-HOPDA, 3-Cl HOPDA, 3-Me HOPDA, and 3,10-diF HOPDA were enzymatically generated from DHB, 4-Cl DHB, 4-Me DHB, and 4,4Ј-diF DHB, respectively, using DHBD as previously described (8,20). The preparation of DHB and chlorinated DHBs has been described elsewhere (29).…”

Section: Methodsmentioning

confidence: 99%

“…The 10-fluoro substituent is not expected to greatly affect catalysis because HOPDAs with small, electronwithdrawing substituents (e.g. -Cl and -CF 3 ) at this position are hydrolyzed by BphD with k cat values within 25% of that for HOPDA (8,28). Significantly, reduction of the volume of the electronegative 3-substituent by 40% upon chlorine-to-fluorine substitution resulted in a 150-fold increase in k cat ( Table 1), indicating that larger 3-substituents interfere with catalysis.…”

Section: Steady-state Kinetics-to Assess the Basis Of Inhibition Ofmentioning

confidence: 99%

“…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 (t1 ⁄ 2 ϳ 500 h) is more stable than 4-Cl HOPDA, which undergoes a non-enzymatic transformation to 4-OH HOPDA (t1 ⁄ 2 ϭ 2.8 h) followed by degradation to products that include acetophenone (t1 ⁄ 2 ϳ 180 h) (8). Second, 3-Cl HOPDA is a very poor substrate of BphD LB400 , effectively inhibiting the hydrolysis of HOPDA (K ic ϭ 0.57 M) more potently than either 4-Cl HOPDA (K ic ϭ 3.6 M) or 4-OH HOPDA (K ic ϭ 0.95 M).…”

mentioning

confidence: 99%

“…For example, 2Ј,6Ј-diCl 2,3-dihydroxybiphenyl (2Ј,6Ј-diCl-DHB) inhibits the third pathway enzyme, DHB dioxygenase (DHBD) in the potent PCB-degrading strain Burkholderia xenovorans LB400 (7). 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 (8) and Rhodococcus globerulus P6 (8 -10) as described below. Interestingly, a glutathione S-transferase in B. xenovorans LB400, BphK, catalyzes the dehalogenation of 3-Cl HOPDA (11) and DxnB2, a homolog of BphD from the dibenzofuran catabolic pathway of Sphingomonas wittichii RW1, catalyzes the hydrolysis of 3-Cl HOPDA (8 -10).…”

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confidence: 99%

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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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Characterization of an extradiol dioxygenase involved in the catabolism of lignin‐derived biphenyl

et al. 2017

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In the catabolism of lignin-derived biphenyl by Sphingobium sp. SYK-6, LigZ catalyzes the cleavage of 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) to a meta-cleavage product (MCP) identified here as 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (DCHM-HOPDA). DCHM-HOPDA is transformed nonenzymatically, likely to a lactone (k = 0.13 ± 0.01 min , pH 7.5). This is hydrolyzed to the dienolate at alkaline pH (apparent pK ~ 11.3). Only the dienolate is a substrate for LigY, the putative MCP hydrolase. LigZ has higher specificity for OH-DDVA (k /K = 2.20 ± 0.02 × 10 s ·m ) than for protocatechuate (PCA; 6 ± 1 × 10 s ·m ). PCA also inactivates LigZ (partition ratio of 50), but at rates too low to be physiologically relevant. This study provides insight into the bacterial catabolism of lignin and facilitates the study of downstream catabolic enzymes.

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

Cited by 93 publications

References 33 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

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

Characterization of an extradiol dioxygenase involved in the catabolism of lignin‐derived biphenyl

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