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

Abstract

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...

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

“…This species very slowly decayed (1/ 2 ϭ 0.0077 s Ϫ1 ) at a rate that matches the previously measured k cat value (Table 1). Interestingly, this rate is similar to that of tautomerization of HOPDA in solution as measured by deuterium exchange experiments (20,22). The absence of E⅐S red accumulation suggests that 3-Cl HOPDA tautomerization is slower than hydrolysis.…”

“…In a previous single turnover ([E] Ͼ [S]) stopped-flow experiment using WT BphD and HOPDA, we observed rapid (ϳ500 s Ϫ1 ) formation of E⅐S red ( max ϭ 492 nm) from the free HOPDA enolate ( max ϭ 434 nm) (Fig. 2E) (20). A similar E⅐S red intermediate ( max ϭ 506 nm) was rapidly formed (ϳ500 s Ϫ1 ) and trapped in the hydrolytically impaired S112A variant (Fig.…”

“…5. Unsubstituted HOPDA is rapidly converted to E⅐S red under single turnover conditions (1/ 1 ϳ 500 s Ϫ1 ), and the presence of an isosbestic point at ϳ460 nm suggests direct transformation (19,20). Large 3-substitu- FIGURE 5.…”

“…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).…”

“…Crystallographic data for the E⅐S red intermediate trapped in the S112A⅐HOPDA complex are most consistent with the bound HOPDA being ketonized (19), 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 (24 -28) or an acyl-enzyme intermediate (19,20). As noted above, HOPDAs that are chlorinated on the dienoate moiety are poorly transformed by BphD (8 -10).…”