Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase

Abstract

The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λmax at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λmax at 437 nm. The time course of formation/depletion of the PKB–ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB–ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λmax at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 Å) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λmax at 473 nm to the PAA–ThDP adduct. The structure of the PAA–ThDP covalent complex is the first example of a product–ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

Catalysis in Enzymatic Decarboxylations: Comparison of Selected Cofactor-Dependent and Cofactor-Independent Examples.

“…While BFDC converts benzoylformate to benzaldehyde, the enzyme also catalyzes a benzoin-type condensation of benzaldehyde in the reverse reaction, similar to the reverse reaction of BAL. When reacting the benzaldehyde product with BFDC, there appeared an absorbance (and a CD band) at 390 nm, in the wavelength region predicted by models 63,64 , but no CD band was evident in the 300–310 nm region 58 . Also, when ( R )-benzoin was added to BAL, there was formed the same CD band at 390 nm indicating slow release of the first benzaldehyde, and the stability of the enamine in the forward direction 54 .…”

“…The enamine intermediate derived from benzoylformate (modeled with λ max of 380 nm) 63,64 has been observed directly on the enzyme BFDC at 390 nm 58 . While BFDC converts benzoylformate to benzaldehyde, the enzyme also catalyzes a benzoin-type condensation of benzaldehyde in the reverse reaction, similar to the reverse reaction of BAL.…”

“…With YPDC, BFDC and BAL, the enamine could be observed directly at 430 nm with 3-PKB as alternate substrate on YPDC. 54–58 …”

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

“…The γ-methyl of Leu377 is within <2.5 Å of several carbon atoms of the phenyl ring of MBP, whereas Tyr460 has a potential edge-to-edge contact with MBP that is also within <2.5 Å. While this appears to be at odds with the kinetic data indicating only a modest effect on K m value for benzoylformate, there is structural evidence that wtBFDC is able to accommodate unnaturally large substrates by slight rotations of active site residues [43]. Therefore it is not unreasonable to suggest that the side chains of Tyr460 and Leu377 could rotate to avoid or, at least, ameliorate unfavorable interactions with the MThDP.…”

“…inhibitor and the engineered mutations ( Figure 5 [43]. Therefore it is not unreasonable to suggest that the side chains of Tyr460 and Leu377 could rotate to avoid or, at least, ameliorate unfavorable interactions with the MThDP.…”