Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis
Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis
复制标题
DOI：
10.1074/jbc.m205484200
复制
发表时间：
2003-01-03
影响因子：
4.8
通讯作者：
Doi, Y
中科院分区：
文献类型：
--
作者：
Hisano, T;Tsuge, T;Doi, Y
来自嗜水气单胞菌的（R）-特异性烯酰辅酶A水合酶（（R）-水合酶）催化将水分子添加到链长为4-6个碳的反式-2-烯酰辅酶A（CoA）上，以产生相应的（R）-3-羟酰基-CoA。它形成分子量约为14，000的相同亚基的二聚体，并参与聚羟基链烷酸酯（PHA）的生物合成。该酶的晶体结构已在1.5埃分辨率下测定。单体的结构由五链反平行β-折叠和中心α-螺旋组成，折叠成所谓的“热狗”折叠，具有悬垂片段。该突出端含有保守残基，包括水合酶2基序残基。在二聚体形式中，两个β折叠相关联以形成延伸的10链β折叠，并且突出端掩盖了亚基界面处的推定活性位点。活性位点位于底物结合隧道的深处，其中Asp（31）和HiS（36）形成催化二联体。这些残基是催化重要的，如通过定点诱变证实的，并且可能分别负责水分子的活化和底物分子的质子化。残基如Leu（65）和瓦尔（130）位于底物结合通道的底部，确定了酶对底物链长的偏好。这些结果为蛋白质工程提供了靶残基，这将增强该酶在新型PHA聚合物生产中的重要性。此外，本研究提供了（R）-水合酶家族的第一个结构信息，并可能促进该家族成员的进一步功能研究。
The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-Angstrom resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and HiS(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family.