Probing the active center, of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester
Probing the active center, of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester
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DOI：
10.1021/bi8004413
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发表时间：
2008-07-22
期刊：
影响因子：
2.9
通讯作者：
Ringe, Dagmar
中科院分区：
文献类型：
--
作者：
Brandt, Gabriel S.;Nemeria, Natalia;Ringe, Dagmar
苯甲醛裂解酶（BAL）利用二磷酸硫胺素和Mg 2+作为辅因子催化（R）-安息香可逆裂解为苯甲醛。该酶通过其碳化反应机制对广泛的化合物的化学酶促合成是重要的。除了其主要功能外，BAL还可以使芳香族氨基酸如苯甲酰甲酸缓慢脱羧。它也是有趣的机制，由于缺乏酸碱残基的活性中心，可以参与质子转移步骤被认为是必要的这些类型的反应。苯甲酰膦酸甲酯是苯甲酰甲酸的一种优良的静电类似物，它被用来探讨苯甲醛裂解酶的作用机理。苯甲醛裂解酶在其与苯甲酰膦酸甲酯的共价复合物中的结构被确定为2.49埃（蛋白质数据库条目3D 7 K），并且代表该酶的第一个结构，其中化合物结合在活性位点中。与酶与硫胺素二磷酸的复合物相比，没有检测到大的结构重组。通过结构阐明了二羧化硫胺素结合中间体的构型。光谱和X-射线结构的研究是一致的抑制所产生的结合MBP的硫胺素二磷酸在活性中心。我们还描绘了His 29（除了高度保守的Glu 50之外，活性位点中唯一潜在的酸碱催化剂）和Trp 163在辅因子活化和苯甲醛裂解酶催化中的作用。
Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 angstrom (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.