Kinetic and Stereochemical Analysis of YwhB, a 4-Oxalocrotonate Tautomerase Homologue in Bacillus subtilis:  Mechanistic Implications for the YwhB- and 4-Oxalocrotonate Tautomerase-Catalyzed Reactions

Abstract

YwhB, a 4-oxalocrotonate tautomerase (4-OT) homologue in Bacillus subtilis, has no known biological role and the gene has no apparent genomic context. The kinetic and stereochemical properties of YwhB have been examined using available enol and dienol compounds. The kinetic analysis shows that YwhB has a relatively non-specific 1,3-and 1,5-keto-enol tautomerase activity, with the former activity prevailing. Replacement of Pro-1 or Arg-11 with an alanine significantly reduces or abolishes these activities, implicating both residues as critical ones for the activities. In D 2 O, ketonization of two monoacid substrates (2-hydroxy-2,4-pentadienoate and phenylenolpyruvate) produces a mixture of stereoisomers {2-keto-3-[ 2 H]-4-pentenoate and 3-[ 2 H]-phenylpyruvate}, where the (3R)-isomers predominate. Ketonization of 2-hydroxy-2,4-hexadienedioate, a diacid, in D 2 O affords mostly the opposite enantiomer, (3S)-2-oxo-[3-2H]-4-hexenedioate. The mono-and diacids apparently bind in different orientations in the active site of YwhB, but the highly stereoselective nature of the YwhB reaction using a diacid suggests that the biological substrate for YwhB may be a diacid. Moreover, of the three dienols examined, 1,3-and 1,5-keto-enol tautomerization reactions are only observed for 2-hydroxy-2,4-hexadienedioate, indicating that the C-3 and C-5 positions are accessible for protonation in this compound. Incubation of 4-OT with 2-hydroxy-2,4-hexadienedioate in D 2 O results in a racemic mixture of 2-oxo-[3-2 H]-4-hexenedioate, suggesting that 4-OT may not catalyze a 1,3-keto-enol tautomerization reaction using this dienol. It has previously been shown that 4-OT catalyzes the near stereospecific conversion of 2-hydroxy-2,4-hexadienedioate to (5S)-[5-2 H]-2-oxo-3-hexenedioate in D 2 O. Taken together, these observations suggest that 4-OT might function as a 1,5-keto-enol tautomerase using 2-hydroxy-2,4-hexadienedioate.4-Oxalocrotonate tautomerase (4-OT) is encoded by the TOL plasmid pWW0, which is present in various soil bacteria including Pseudomonas putida mt-2 (1,2). The enzyme is part of the meta-fission pathway, which is a catabolic route for the conversion of simple aromatic hydrocarbons to Krebs Cycle intermediates (3). The hexameric enzyme, composed of 62 amino † This research was supported by the National Institutes of Health Grants GM-65324 and GM-41239 and the Robert A. Welch Foundation (F-1334). S.C.W. is a Fellow of the American Foundation for Pharmaceutical Education.*Address correspondence to this author. Tel: 512-471-6198; Fax: 512-232-2606; E-mail: whitman@mail.utexas acid monomers, purportedly catalyzes the conversion of 2-oxo-4-hexenedioate (1,Scheme 1) to 2-oxo-3-hexenedioate (3) via the intermediate 2-hydroxy-2,4-hexadienedioate, known commonly as 2-hydroxymuconate (2) (4-7). This reaction, a 1,3-allylic rearrangement, was proposed on the basis of indirect kinetic and stereochemical studies using 2 and other monoand diacid dienols (4,6,7). The conversion of 1 to 3 cannot be observed directly because 1...

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

“…The ketonization of 1 to 2 was measured by following the increase in absorbance at 236 nm (ε = 6580 M −1 cm −1 ) using substrate concentrations ranging from 10–200 μM (4). The ketonization of 3 to 4 was measured by following the decrease in absorbance at 283 nm (ε = 18,000 M −1 cm −1 ) using substrate concentrations ranging from 10–140 μM (5). An aliquot of enzyme was diluted into the potassium phosphate buffer, yielding a final dimer concentration of 1–191 nM for reactions using 1 and 100–1000 nM for reactions using 3 .…”

Bridging between Organocatalysis and Biocatalysis: Asymmetric Addition of Acetaldehyde to β‐Nitrostyrenes Catalyzed by a Promiscuous Proline‐Based Tautomerase

“…Residue Pro-1 functions as a general base (pK a % 6.4) that transfers the 2-hydroxy proton of 1 to the C5-position to give 2. [14] Residues Arg-11 and Arg-39 are important for binding of 1 and interact with the C6 and C1 carboxylate groups of 1, respectively. [15] 4-OT also accepts phenylenolpyruvate (3) and p-hydroxyphenylenolpyruvate (5) as substrates for tautomerization, giving phenylpyruvate (4) and p-hydroxyphenylpyruvate (6) as products, respectively (Scheme 1).…”

The kinetic characterization and X-ray structure of a putative benzoylformate decarboxylase from M. smegmatis highlights the difficulties in the functional annotation of ThDP-dependent enzymes

“…However, the gene directly downstream from that of MsBFDC (MSMEG_1607) was predicted to encode a 4-oxalocrotonate tautomerase (4-OT). It is notable that both BFDC and 4-OT are involved in the degradation of aromatic compounds [17,58] and, in addition, 4-OT also catalyzes the formation of several 2-keto acids [59]. Since the known BFDCs are found in operons (and MsBFDC is not), it was conceivable that this putative 4-OT might be the source of MsBFDC's substrate.…”