Crystal Structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8
Crystal Structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8
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DOI：
10.1074/jbc.m703440200
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发表时间：
2007-11-09
影响因子：
4.8
通讯作者：
Miki, Kunio
中科院分区：
文献类型：
--
作者：
Kim, Seong-Hoon;Hisano, Tamao;Miki, Kunio
4-羟基苯乙酸（4 HPA）3-单加氧酶参与4 HPA降解途径的初始步骤，并催化4 HPA羟基化为3，4-二羟基苯乙酸。这种酶由两种组分组成，加氧酶（HpaB）和还原酶（HpaC）。为了理解HpaB催化机制的结构基础，在三种状态下测定了来自嗜热栖热菌HB 8的HpaB的晶体结构：无配体形式、与FAD的二元复合物以及与FAD和4 HPA的三元复合物。结构分析表明，黄素的结合和解离伴随着β 5和β 6之间的环和β 8和β 9之间的环的构象变化，导致部分底物结合位点（Ser-197和Thr-198）的预形成。后一个环在结合4 HPA后进一步改变其构象，并阻碍活性位点与本体溶剂的接触。Arg-100位于推定的氧结合位点附近，并且可能参与C4-ahydroperoxoreflavin中间体的形成和稳定。
The 4-hydroxyphenylacetate ( 4HPA) 3-monooxygenase is involved in the initial step of the 4HPA degradation pathway and catalyzes 4HPA hydroxylation to 3,4-dihydroxyphenylacetate. This enzyme consists of two components, an oxygenase ( HpaB) and a reductase ( HpaC). To understand the structural basis of the catalytic mechanism of HpaB, crystal structures of HpaB from Thermus thermophilus HB8 were determined in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4HPA. Structural analysis revealed that the binding and dissociation of flavin are accompanied by conformational changes of the loop between beta 5 and beta 6 and of the loop between beta 8 and beta 9, leading to preformation of part of the substrate-binding site ( Ser-197 and Thr-198). The latter loop further changes its conformation upon binding of 4HPA and obstructs the active site from the bulk solvent. Arg-100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4-ahydroperoxyflavin intermediate.