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
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
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DOI：
10.1016/j.bbapap.2015.04.027
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发表时间：
2015-08-01
影响因子：
3.2
通讯作者：
McLeish, Michael J.
中科院分区：
文献类型：
--
作者：
Andrews, Forest H.;Horton, Joshua D.;McLeish, Michael J.
苯甲酰甲酸脱羧酶 (BFDC) 是一种二磷酸硫胺素 (ThDP) 依赖性酶，可催化苯甲酰甲酸的非氧化脱羧。它是扁桃酸途径和D-苯基甘氨酸降解途径中的倒数第二个酶。 ThDP 依赖性酶工程数据库 (TEED) 现在列出了 800 多个注释为 BFDC 的序列，其中包括来自耻垢分枝杆菌 (MsBFDC) 的一个序列。然而，没有证据表明耻垢分枝杆菌基因组中存在任何苯甲酰甲酸形成途径。此外，MsBFDC 与从恶臭假单胞菌 (PpBFDC) 中分离出来的充分表征的酶的序列比对表明，MsBFDC 中存在活性位点取代，可能会降低苯甲酰甲酸的活性。综合这些数据表明注释不太可能是正确的。为了测试这一假设，对假定的 MsBFDC 进行了克隆、表达、纯化，并将 X 射线结构解算至分辨率为 2.2 埃。虽然没有显示活性位点存在 ThDP 的证据，但其结构与 PpBFDC 的结构非常相似。测试了多种 2-含氧酸作为底物。对于 MsBFDC，苯甲酰甲酸的 K-m 值类似于 23 mM，比 PpBFDC 高近 100 倍，而 k(cat) 值降低了 60 倍。这些值表明苯甲酰甲酸不是该酶的生理底物，并且注释为 2-含氧酸脱羧酶可能更合适。 (C) 2015 Elsevier B.V. 保留所有权利。
Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the nonoxidative decarboxylation of benzoylformate. It is the penultimate enzyme in both the mandelate pathway and the D-phenylglycine degradation pathway. The ThDP-dependent Enzyme Engineering Database (TEED) now lists more than 800 sequences annotated as BFDCs, including one from Mycobacterium smegmatis (MsBFDC). However, there is no evidence that either pathway for benzoylformate formation exists in the M. smegmatis genome. Further, sequence alignments of MsBFDC with the well characterized enzyme isolated from Pseudomonas putida (PpBFDC) indicate that there will be active site substitutions in MsBFDC likely to reduce activity with benzoylformate. Taken together these data would suggest that the annotation is unlikely to be correct. To test this hypothesis the putative MsBFDC was cloned, expressed, purified, and the X-ray structure was solved to a resolution of 2.2 angstrom. While showing no evidence for ThDP in the active site, the structure was very similar to that of PpBFDC. A number of 2-oxo acids were tested as substrates. For MsBFDC the K-m value for benzoylformate was similar to 23 mM, nearly 100-fold greater than that of PpBFDC while the k(cat) value was reduced 60-fold. These values would suggest that benzoylformate is not the physiological substrate for this enzyme, and that annotation as a 2-oxo acid decarboxylase may be more appropriate. (C) 2015 Elsevier B.V. All rights reserved.