Snapshot of a reaction intermediate: analysis of benzoylformate decarboxylase in complex with a benzoylphosphonate inhibitor

Abstract

Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate- (ThDP-) dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereo control in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral alpha-hydroxy ketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2alpha-phosphonomandelyl-ThDP) of the covalent ThDP-substrate adduct C2alpha-mandelyl-ThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates) and by high-resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography to a spatial resolution of 1.37 A (PDB ID 3FSJ). The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of real-time kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.

Figure 1

Concentration dependence of inhibition of BFDC by MBP. The MBP concentration was held at (●) 0, (○) 0.25, (▼) 0.5, (◊) 0.75, (■) 1.25 and (□) 2 mM while the benzoylformate concentration was varied between (0.3–3×K_m).

Figure 2

Circular dichroism studies of the binding of MBP to BFDC. a. Difference CD spectra showing formation of 1’,4’-imino PMThDP on BFDC. Conditions are described in Materials and methods. b. Changes in ellipticity at 299 nm with increasing concentration of MBP in Figure 2A. c. Rate of 1’,4’-imino PMThDP formation monitored on stopped-flow CD when BFDC (66.4 µM active site concentration) in 50 mM KH₂PO₄ (pH 6.0) was mixed with an equal volume of 4 mM MBP in the same buffer at 30 °C. Reaction was monitored for 5 s at 302 nm with a path length of 1 cm and slit width of 2 nm. A total of 5000 data points were collected at 5 ms intervals.

Figure 3

Circular dichroism titration of BFDC with MAP. a. Difference spectra of BFDC (1.7 mg/ml, concentration of active centers = 30.2 µM) on addition of 1–30 mM MAP. The conditions of the experiments are described in Materials and methods. Inset. Dependence of the ellipticity at 299 nm on concentration of MAP. b. The reversibility of 1’, 4’-iminophosphonolactyl ThDP formation on BFDC: 1- baseline recorded with 10 mM KH₂PO₄ (pH 6.0); 2- BFDC (1.70 mg/ml) in the presence of 2.50 mM MgCl₂ and 0.50 mM ThDP; 3- BFDC as in line 2 with 30 mM MAP; 4- as in line 3 after overnight incubation at 4 °C; 5-as in line 4 after 3 hours of dialysis against 2L of 20 mM KH₂PO₄ (pH 6.0).

Figure 4

Evidence for the formation of a covalent adduct between the cofactor ThDP and the mechanism-based inhibitor MBP by FT-ICR mass spectrometry. Cofactor adduct was identified from reaction mixture subsequent to TCA precipitation of protein.

Figure 5

Electron density maps (grey mesh) for the BFDC-bound MBP-ThDP adduct, demonstrating the occupancy of the inhibitor phenyl ring and confirming the presence of electron density resulting from the newly formed bond. The indicated electron density results from a simulated annealing-refined 2Fo - Fc omit map contoured to 0.9 σ.

Figure 6

Stereo view of binding geometry of MBP-ThDP adduct of benzoylformate decarboxylase (BFDC), showing likely hydrogen bonds between enzyme and adduct.

Figure 7

Comparison of the inhibitor MBP bound in the active sites of benzoylformate decarboxylase (BFDC, a) and benzaldehyde lyase (BAL, b).

Figure 8

Possible conformations for ThDP-bound HPP intermediate, based on the observed (S) stereochemistry of the 2-HPP product. a. Schematic of available conformations for HPP intermediate. b. Active site of BFDC with MBP. Likely hydrogen bonds indicated in orange. Heavy atom distances to nearest atoms of residues making up the methyl binding pocket shown in yellow. c. Active site of BFDC with predicted geometry of ThDP-bound HPP intermediate model.

Figure 9

Analysis of intermediate binding to the BAL active site. a. Predicted geometry of ThDP-bound HPP intermediate for BAL, based on observed (R) stereochemistry of 2-HPP product and favorable interaction with His29-positioned water. b. View of the BAL active site with MBP bound. Note that schematic and surface views are rotated ~90° relative to each other. c. View of BAL with predicted ThDP-bound HPP intermediate.

Scheme 1

Activity of benzoylformate decarboxylase (BFDC) toward benzoin (a), benzaldehyde (b) and acetaldehyde with benzaldehyde (c).

Scheme 2

Mechanism of formation of the pre-decarboxylation intermediate from benzoylformate or MBP.

Scheme 3

Schematic of mechanism of BFDC-catalyzed HPP formation from benzaldehyde and acetaldehyde (a) and relationship (b) of ThDP-MBP complex to penultimate intermediate (boxed at right).