Crystal structure of thiaminase-I from Bacillus thiaminolyticus at 2.0 Å resolution
Crystal structure of thiaminase-I from Bacillus thiaminolyticus at 2.0 Å resolution
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DOI：
10.1021/bi981673l
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发表时间：
1998-11-10
期刊：
影响因子：
2.9
通讯作者：
Ealick, SE
中科院分区：
文献类型：
--
作者：
Campobasso, N;Costello, CA;Ealick, SE
硫胺素酶-I催化硫胺素的噻唑部分被各种亲核试剂如吡啶、苯胺、儿茶酚、喹啉和半胱氨酸取代。来自解硫胺芽孢杆菌的酶的晶体结构在2.5埃分辨率下通过多个同晶置换测定，并精确到R因子0.195（无R = 0.272）。另外两种结构，一种是天然的，一种含有共价结合的抑制剂，通过从第二种晶体形式进行分子置换在2.0埃分辨率下测定，并分别精确到0.205和0.217的R因子（R-free = 0.255和0.263）。整体结构包含由大裂缝分开的两个α/β型结构域。在裂缝的底部是Cys 113，以前被确定为一个关键的活性位点亲核试剂。具有共价结合的硫胺素类似物的结构，其作为基于机制的灭活剂起作用，证实了活性位点的位置。Glu 241似乎作为一个活性位点的基地，以增加Cys 113的亲核性。制备了突变体Glu 241 Gln，其没有显示出活性。硫胺素酶-I与序列数据库中的其他蛋白质没有序列同一性，但其三维结构与周质结合蛋白和转铁蛋白具有非常高的结构同源性。
Thiaminase-I catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles, such as pyridine, aniline, catechols, quinoline, and cysteine. The crystal structure of the enzyme from Bacillus thiaminolyticus was determined at 2.5 Angstrom resolution by multiple isomorphous replacement and refined to an R factor of 0.195 (R-free = 0.272). Two other structures, one native and one containing a covalently bound inhibitor, were determined at 2.0 Angstrom resolution by molecular replacement from a second crystal form and were refined to R factors of 0.205 and 0.217 (R-free = 0.255 and 0.263), respectively. The overall structure contains two alpha/beta-type domains separated by a large cleft. At the base of the cleft lies Cys113, previously identified as a key active site nucleophile. The structure with a covalently bound thiamin analogue, which functions as a mechanism-based inactivating agent, confirms the location of the active site. Glu241 appears to function as an active site base to increase the nucleophilicity of Cys113. The mutant Glu241Gln was made and shows no activity. Thiaminase-I shows no sequence identity to other proteins in the sequence databases, but the three-dimensional structure shows very high structural homology to the periplasmic binding proteins and the transferrins.