Pyruvate Decarboxylase:  A Molecular Modeling Study of Pyruvate Decarboxylation and Acyloin Formation

Abstract

Using crystal structure data for the pyruvate decarboxylase from Saccharomyces uvarum (which is nearly identical with the enzyme from Saccharomyces cerevisiae), molecular modeling studies have been carried out to investigate the mode of action of the enzyme. Each step of the decarboxylation mechanism can be explained by assuming that the 4‘-amino group of thiamin diphosphate (TDP) acts as a general acid and, in its deprotonated form, as a general base. The carboxyl group of Glu 477 plays a key role in both pyruvate decarboxylation and acyloin formation. In the first case it interacts with the carboxylate group of pyruvate to stabilize the incipient dianion formed by attack of the thiazolium carbanion on pyruvate. In the second case, it interacts with the developing alkoxide anion arising from attack on acetaldehyde or benzaldehyde by the carbanion−enamine intermediate. These studies have permitted the assignment of configuration to all of the key intermediates in the catalytic process. Thus the carbanion−enamine intermediate 5 is found to have the E-configuration. The S-configuration is imposed on the 2-(2-hydroxypropionyl)ethyl)thiamin diphosphate intermediate 4 by the chiral conformation induced in the achiral cofactor through its interactions with the protein. The R-configuration is assigned to the 2-(1-hydroxyethyl)thiamin diphosphate intermediate 6 arising through protonation of the carbanion−enamine intermediate 5. The tight stereochemical control observed in acyloin formation from aromatic aldehydes and pyruvate is explained, as is the relaxed stereocontrol in acyloin formation from acetaldehyde and pyruvate.

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

“…The conformation of the carbanion͞enamine intermediate with respect to the thiazolium ring, the C␣, C␤, and the C␣ oxygen atoms of the ␣,␤ dihydroxyethyl moiety observed in TK is very likely common to other ThDP-dependent enzymes. For instance, it agrees well with the conformation of the hydroxyethyl ThDP in pyruvate decarboxylase derived from modeling studies (26). The mechanistic conclusions concerning the 4-NH 2 group of ThDP, therefore, may hold also for other ThDP-dependent enzymes.…”

“…These mechanistic differences are reflected in the activesite topology of the two enzymes. In pyruvate decarboxylase, there is a residue (Glu-477) suitably positioned to participate in proton transfer (26), whereas in TK, no such amino acid is found close to the C␣ atom of the intermediate.…”

“…Attempts to model the intermediate with sp 3 geometry at the ␣ carbon atom are not consistent with the electron density and also result in a small but significant increase of the free R factor by 0.4%. The carbanion͞ enamine has the E-configuration, as predicted from molecular modeling of decarboxylation of pyruvate in pyruvate decarboxylase (26). The ␣,␤ dihydroxyethyl moiety is held in place through a number of hydrogen bonds made to surrounding amino acids (Fig.…”

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

“…This conclusion is reached since PDHc-E1 preferentially synthesized (S)-acetoin and (R)-acetolactate, while YPDC formed (R)-acetoin and (S)-acetolactate. That YPDC and pyruvate decarboxylase from Zymomonas mobilis produce opposite enantiomers of acetoin had been noted earlier, and Lobell and Crout carried out molecular modeling using the 3-dimensional X-ray structure of YPDC to provide an explanation for its selectivity in acetoin formation [23].…”

Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of Azospirillum brasilense

“…Glu468 is a conserved catalytic residue in pyruvate decarboxylase and IPDC Ec , which has been proposed to be involved in both preand postdecarboxylation steps. More specifically a role in protonation of the enamine-carbanion intermediate has been proposed (20,26,32). Enzymes with amino acid substitutions in this position showed an impaired release of the aldehyde product.…”