The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.
The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.
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DOI：
10.1046/j.1432-1033.2002.03151.x
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发表时间：
2002-09
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通讯作者：
M. Yamanishi;Michio Yunoki;T. Tobimatsu;Hideaki Sato;J. Matsui;Ayako Dokiya;Yasuhiro Iuchi;Kazunori Oe;K. Suto;N. Shibata;Y. Morimoto;N. Yasuoka;T. Toraya
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作者：
M. Yamanishi;Michio Yunoki;T. Tobimatsu;Hideaki Sato;J. Matsui;Ayako Dokiya;Yasuhiro Iuchi;Kazunori Oe;K. Suto;N. Shibata;Y. Morimoto;N. Yasuoka;T. Toraya
将重组肺炎克雷伯氏菌甘油脱氢酶纯化至均一。该酶的亚基组成很可能是α 2 β 2 γ 2。当（R）-和（S）-丙烷-1，2-二醇单独用作底物时，（R）-对映体的速率比（S）-异构体快2.5倍。与二醇脱氢酶（一种同功能酶）相反，该酶对（S）-异构体的亲和力基本上与对（R）-异构体的亲和力相同或仅略高于对（R）-异构体的亲和力（Km（R）/Km（S）= 1.5）。在2.1 A分辨率下测定了甘油脱氢酶与氰钴胺和丙二醇复合物的晶体结构。该酶以α β γ异源三聚体的二聚体形式存在。钴胺素以所谓的“碱基对”模式结合在α和β亚基之间的界面处，其中核苷酸部分的5，6_二甲基苯并咪唑与钴原子配位。氰基的电子密度几乎是不可观察的，这表明氰钴胺被还原为钴（II）维生素通过X-射线照射。活性位点在由α亚基的中心区域形成的（β/α）8桶中。底物丙烷-1，2-二醇和必需辅因子K+结合在钴胺素的咕啉环上方的（β/α）8桶内。K+由底物的两个羟基和活性位点残基的五个氧原子七配位。这些结构特征与二醇脱氢酶的结构特征非常相似。甘油二酯酶中α和β亚基之间的紧密接触可能使人联想到该酶对腺苷钴胺素的亲和力高于二醇二酯酶。尽管外消旋丙烷-1，2-二醇用于结晶，但与甘油脱氢酶结合的底物被分配给（R）-异构体。这与二醇脱氢酶形成鲜明对比，并解释了两种酶对甘油自杀失活的敏感性差异。
Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.