Campobasso N, Costello C A, Kinsland C, Begley T P, Ealick S E
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 1998 Nov 10;37(45):15981-9. doi: 10.1021/bi981673l.
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 A resolution by multiple isomorphous replacement and refined to an R factor of 0.195 (Rfree = 0.272). Two other structures, one native and one containing a covalently bound inhibitor, were determined at 2.0 A resolution by molecular replacement from a second crystal form and were refined to R factors of 0.205 and 0.217 (Rfree = 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.
硫胺素酶-I催化硫胺素的噻唑部分被多种亲核试剂取代，如吡啶、苯胺、儿茶酚、喹啉和半胱氨酸。通过多同晶置换法以2.5埃的分辨率测定了硫胺素分解芽孢杆菌中该酶的晶体结构，并将其精修至R因子为0.195（Rfree = 0.272）。通过从第二种晶体形式进行分子置换，以2.0埃的分辨率测定了另外两种结构，一种是天然结构，另一种含有共价结合的抑制剂，分别精修至R因子为0.205和0.217（Rfree = 0.255和0.263）。整体结构包含两个由大裂缝隔开的α/β型结构域。在裂缝底部是Cys113，先前被确定为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，其作为基于机制的失活剂，证实了活性位点的位置。Glu241似乎作为活性位点碱基来增加Cys113的亲核性。制备了突变体Glu241Gln，其无活性。硫胺素酶-I在序列数据库中与其他蛋白质没有序列同一性，但三维结构显示与周质结合蛋白和转铁蛋白具有非常高的结构同源性。
