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

Abstract

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21473-21482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 activecenter variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…Earlier, 1 H NMR and LC-MS analyses of this reaction assigned the product as 2-hydroxy-3-oxoadipate, which is decarboxylated non-enzymatically to hydroxylevulinate (7). By analogy with carboligase reactions of other ThDP-dependent enzymes (16,17), we assigned the observed band to the (R)-HOA enantiomer. The progress curves measured at 278 nm allowed determination of the steady state linear velocities of HOAS-catalyzed production of (R)-HOA (Fig.…”

A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

“…Because Gln-408 is not involved in any major interactions other than the one with the intermediate analog through water, it is unlikely that the unusual carboligation results with this variant are because of loop disorder. This is particularly interesting because other important amino acids such as Glu-636 and Tyr-177 also interact with the intermediate analog through water, and their substitution also caused remarkable changes in the biochemical behavior of enzyme (15,18). The E636A substitution converted the enzyme to an acetolactate synthase with very high stereoselectivity, which, based on Fourier transform ion cyclotron resonance mass spectrometry of tryptic digest of variant and E1ec, could be attributed again to changes in outer loop dynamics.…”

“…Chiral Gas Chromatography-To analyze the carboligation side products and their enantiomeric excess, chiral gas chromatography (GC) was used (18). The carboligation reaction was initiated by adding 8.0 mM pyruvate to 10.0 mM KH 2 PO 4 (pH 7.0) containing 0.2 mM ThDP, 1.0 mM Mg 2ϩ , and 30.0 M E1ec or its variants and incubated overnight at 4°C.…”

Investigation of the donor and acceptor range for chiral carboligation catalyzed by the E1 component of the 2-oxoglutarate dehydrogenase complex

“…Hence, the ( S )-enantiomer has a longer retention time. [9] By using the same method racemic benzoin and ( R ) benzoin were used as standards (data not shown).…”

“…[8] The Rutgers group has previously constructed several active site variants in yeast pyruvate decarboxylase (YPDC) from Saccharomyces cerevisiae and in the E1 component of the Escherichia coli pyruvate dehydrogenase complex (E1p) which were capable of catalyzing such reactions. [9] The E477Q YPDC variant was an effective acetoin synthase, while the D28A or D28N YPDC variants catalyze acetolactate formation. [10] On the other hand, the E636Q and E636A E1p active site variants also became acetolactate synthases[11].…”