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 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into 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/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately 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/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(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 (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded 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.

Figure 1

The aerobic microbial degradation of biphenyl by the upper Bph pathway is typical of aromatic compound degradation via the meta-cleavage pathway. The meta-cleavage product, HOPDA, is hydrolyzed in an unusual reaction by the final enzyme, BphD.

Figure 2

The proposed enol-keto tautomerization and subsequent hydrolytic C-C cleavage catalyzed by the MCP hydrolases.

Scheme 1

Figure 3

A representative stopped-flow experiment illustrating a single turnover of HOPDA (4 μM) by BphD_LB400 (8 μM) at 3.2 °C in potassium phosphate buffer supplemented with 20% glycerol (I = 100 mM), pH 7.5. A) Time-resolved spectral changes show free HOPDA (λ_max = 434 nm) transformed to an enzyme-bound form, E:S (λ_max = 492 nm). B) Absorbance at 492 nm versus time shows the decay of the E:S intermediate. The solid line denotes the double exponential fit. The inset shows the single exponential fit for the formation of the E:S intermediate. C) Absorbance at 270 nm versus time for the formation/decay of the HPD product. Data points are shown together with the fit of a single exponential (solid line) describing HPD decay. Inset shows the formation of HPD and its fit to a double exponential.

Figure 4

Top: The ¹H NMR spectrum of HPD generated from BphD_LB400-catalyzed hydrolysis of HOPDA in 97% D₂O (potassium phosphate, pD = 7.5, I = 5 mM). Peak integration demonstrates ∼10% deuterium incorporation into H-5_Z and almost complete incorporation into H-5_E. Bottom: The ∼10% non-specific deuterium incorporation into H-5_Z is consistent with non-enzymatic exchange prior to enzymatic catalysis.

Figure 5

Cornish-Bowden plots illustrating the type of inhibition of the BphD_LB400-catalyzed hydrolysis of HOPDA by reaction products. (A) Inhibition by benzoate described by competitive inhibition (K_ic = 220 ± 30 μM; K_m = 0.21 ± 0.02 μM; V = 5.9 ± 0.2 U/mg). Reaction rates determined using 0.24 (■), 0.57 (O), 1.1 (◆), 2.2 (△), and 4.6 μM (●) HOPDA. (B) Inhibition by HPD described by mixed inhibition (K_ic = 84 ± 41 μM; K_iu = 120 ± 30 μM; K_m = 0.45 ± 0.08 μM; V = 4.0 ± 0.2 U/mg). Reaction rates were measured using 0.23 (●), 0.56 (□), 0.82 (◆), and 5.9 μM (△) HOPDA. Equations describing competitive, uncompetitive, and mixed inhibition were each fit to the data using the least squares, dynamic weighting options of the LEONORA program, and the type of inhibition determined by comparing the quality of fit based on non-random trends in the residuals. The solid lines represent the best fit parameters of the global fit at each HOPDA concentration. Conditions: potassium phosphate buffer (I = 100 mM), 20% glycerol, pH 7.5, 5 ± 3 °C.

Figure 6

Left: A schematic diagram of BphD_LB400 shown as a tetramer with 222-point group symmetry. The view is down a crystallographic 2-fold axis parallel to the c axis of the crystal. A and B are crystallographically independent: crystallographic 2-fold symmetry transforms A to A' and B to B'. By contrast, A is related to B or B' by non-crystallographic 2-fold symmetry. Right: A ribbon diagram of the BphD_LB400 tetramer viewed in the same direction. The four subunits are colored green, yellow, cyan, and blue. Arrows highlight the close contacts between lid domains of A and B' (and A' and B).

Figure 7

A ribbon diagram of BphD_LB400 subunit A. The lid domain (residues 146−212) is shown at the top, and the key catalytic residues (Asp237, His265 and Ser112) are shown in ball-and-stick representation.

Figure 8

Stereo view of HOPDA bound to the active site of BphD_LB400 S112C mutant. Top: The refined structure is shown along with (2F_o - F_c) exp(iα_c) electron density (1.0 σ). Bottom: Ball-and-stick stereo model of enzyme-substrate interactions. O, N and S atoms are colored in red, blue, and green, respectively. C atoms are colored in brown for the catalytic residues Asp237-His265-Cys112, gray for the substrate, and yellow for the rest of the residues. Potential hydrogen bonds are shown as dotted lines.

Scheme 2

Scheme 3

Figure 9

A possible mechanism of BphD_LB400. The free enzyme (E) binds and ketonizes HOPDA via His265-catalyzed transfer of a proton (H*) to the proS position at C-5, generating E:S^k. Nucleophilic attack on E:S^k generates the first tetrahedral intermediate (Tet₁), the negative charge of which is stabilized by the main chain amide protons of the ‘oxyanion hole’ residues, Met113 and Gly42. Collapse of Tet₁ involves C-C fragmentation onto the re face of the double bond, generating the acyl-enzyme intermediate (E:B), and releasing HPD with the inserted proton at the H-5_E position. His265 activates water to attack at the acyl-enzyme carbonyl, generating the second tetrahedral intermediate (Tet₂), collapse of which releases benzoate and regenerates free enzyme. For clarity, the conserved residues Asp237 (of the catalytic triad) and Asn51 (interacts with the HOPDA carboxylate) are omitted.