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.
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 nonspecific 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 D2O, ketonization of two monoacid substrates (2-hydroxy-2,4-pentadienoate and phenylenolpyruvate) produces a mixture of stereoisomers {2-keto-3-[2H]-4-pentenoate and 3-[2H]-phenylpyruvate}, where the (3R)-isomers predominate. Ketonization of 2-hydroxy-2,4-hexadienedioate, a diacid, in D2O 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 D2O results in a racemic mixture of 2-oxo-[3-2H]-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)-[5-2H]-2-oxo-3-hexenedioate in D2O. Taken together, these observations suggest that 4-OT might function as a 1,5-keto-enol tautomerase using 2-hydroxy-2,4-hexadienedioate.
Wang
Susan C
SC
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Johnson
William H
WH
Czerwinski
Robert M
RM
Stamps
Stacy L
SL
Whitman
Christian P
CP
D000409:Alanine / Q000378:metabolism; D000473:Alkanes / Q000737:chemistry* / Q000494:pharmacology; D000480:Alkynes / Q000737:chemistry* / Q000494:pharmacology; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002021:Buffers; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010710:Phosphates / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D000473:Alkanes; D000480:Alkynes; D002021:Buffers; D010710:Phosphates; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; D000409:Alanine