Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of beta-amino acids incorporated in secondary metabolites.
Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of beta-amino acids incorporated in secondary metabolites.
复制标题
探索 MIO 依赖性氨基变位酶的活性位点，这是次级代谢产物中 β-氨基酸生物合成的关键催化剂。
DOI：
10.1002/bip.21500
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Bruner,StevenD
中科院分区：
文献类型：
--
作者：
Cooke,HeatherA;Bruner,StevenD
酪氨酸氨基变位酶SgTAM在烯二炔抗肿瘤抗生素C-1027的生物合成中从l-酪氨酸产生（S）-β-酪氨酸。该转化由亚甲基咪唑-5-酮（MIO）辅基促进。MIO首先在氨裂解酶的同源家族中被鉴定，其将芳香族氨基酸脱氨基以形成α，β-不饱和羧酸。裂解酶的底物特异性研究已有报道，但改变氨基变位酶的底物特异性的研究还未见报道。此外，尚不清楚是什么结构特性负责催化氨基重新加入α，β-不饱和中间体形成β-氨基酸。试图阐明SgTAM的特异性和机制决定因素也已被证明是困难的，因为它是通过诱变对活性位点的扰动。X-射线共晶结构的SgTAM突变体的催化基地与l-酪氨酸验证重要的底物结合残基以及酶的基础。进一步的诱变表明，去除这些关键的相互作用使酶失活。通过诱变，时间点测定和X-射线晶体学探测的活性的建议结构决定因素揭示了这些残基在保持关键的四级结构特性，有助于催化的复杂作用。
The tyrosine aminomutase SgTAM produces (S)-β-tyrosine from l-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form α,β-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been no reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the α,β-unsaturated intermediates to form β-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray co-crystal structure of the SgTAM mutant of the catalytic base with l-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis.