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.
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.
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DOI：
10.1021/bi701231a
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发表时间：
2007-10
期刊：
影响因子：
2.9
通讯作者：
Susan C Wang;W. Johnson;R. Czerwinski;S. L. Stamps;C. P. Whitman
中科院分区：
文献类型：
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作者：
Susan C Wang;W. Johnson;R. Czerwinski;S. L. Stamps;C. P. Whitman
YwhB是枯草芽孢杆菌中的4-草酰基互换酶(4-OT)同源物，没有已知的生物学功能，也没有明显的基因组背景。用现有的烯醇和二烯醇类化合物考察了YWHB的动力学和立体化学性质。动力学分析表明，YwhB具有相对非特异性的1，3-和1，5-酮-烯醇互变异构酶活性，且前者的活性占优势。用丙氨酸取代Pro-1或Arg-11显著降低或取消这些活性，这意味着这两个残基都是活性的关键。在D2O中，两种单酸底物(2-羟基-2，4-戊二烯酸酯和苯醇式丙酮酸)的酮化反应生成立体异构体的混合物{2-酮-3-[2H]-4-戊烯酸酯和3-[2H]-苯基丙酮酸}，其中(3R)-异构体占主导地位。2-羟基-2，4-己二烯二酸酯是一种二元酸，在D2O中的酮化反应主要生成相反的对映体，即(3S)-2-氧代-[3-2H]-4-己二烯二酸酯。一元酸和二元酸显然在YWHB的活性中心以不同的方向结合，但YWHB使用二元酸反应的高度立体选择性表明YWHB的生物底物可能是二元酸。此外，在所考察的三个二烯醇中，只有2-羟基-2，4-己二烯二酸酯有1，3-和1，5-酮基-烯醇的互变异构化反应，这表明该化合物中的C-3和C-5位置可以质子化。4-OT与2-羟基-2，4-己二烯二酸酯在D2O中反应生成了2-氧代-[3-2H]-4-己二烯二酸酯的外消旋混合物，表明4-OT可能不催化1，3-酮基-烯醇的异构化反应。已有研究表明，4-OT在D2O中催化2-羟基-2，4-己二烯二酸盐近立体选择性转化为(5S)-[5-2H]-2-氧-3-己二烯二酸盐。综上所述，这些观察结果表明，4-OT可能是使用2-羟基-2，4-己二烯二酸酯的1，5-酮基-烯醇互变异构酶。
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.