Crystal Structure of 4-Methyl-5--hydroxyethylthiazole Kinase from Bacillus subtilis at 1.5 Å Resolution
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- 作者：Nino Campobasso ; Irimpan I. Mathews ; Tadhg P. Begley ; and Steven E. Ealick
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：July 11, 2000
- 年：2000
- 卷：39
- 期：27
- 页码：7868 - 7877
- 全文大小：1054K
- 年卷期：v.39,no.27(July 11, 2000)
- ISSN：1520-4995
文摘
4-Methyl-5-
-hydroxyethylthiazole kinase (ThiK) catalyzes the phosphorylation of the hydroxylgroup of 4-methyl-5--hydroxyethylthiazole (Thz). This enzyme is a salvage enzyme in the thiaminbiosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from1-deoxy-D-xylulose-5-phosphate, cysteine, and tyrosine. The structure of ThiK in the rhombohedral crystalform has been determined to 1.5 Å resolution and refined to a final R-factor of 21.6% (R-free 25.1%).The structures of the enzyme/Thz complex and the enzyme/Thz-phosphate/ATP complex have also beendetermined. ThiK is a trimer of identical subunits. Each subunit contains a large nine-stranded central-sheet flanked by helices. The overall fold is similar to that of ribokinase and adenosine kinase, althoughsequence similarity is not immediately apparent. The area of greatest similarity occurs in the ATP-bindingsite where several key residues are highly conserved. Unlike adenosine kinase and ribokinase, in whichthe active site is located between two domains within a single subunit, the ThiK active site it formed atthe interface between two subunits within the trimer. The structure of the enzyme/ATP/Thz-phosphatecomplex suggests that phosphate transfer occurs by an inline mechanism. Although this mechanism issimilar to that proposed for both ribokinase and adenosine kinase, ThiK lacks an absolutely conservedAsp thought to be important for catalysis in the other two enzymes. Instead, ThiK has a conserved cysteine(Cys198) in this position. When this Cys is mutated to Asp, the enzymatic activity increases 10-fold.Further sequence analysis suggests that another thiamin biosynthetic enzyme (ThiD), which catalyzes theformation of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by two sequential phosphorylation reactions, belongs to the same family of small molecule kinases.
-hydroxyethylthiazole kinase (ThiK) catalyzes the phosphorylation of the hydroxylgroup of 4-methyl-5--hydroxyethylthiazole (Thz). This enzyme is a salvage enzyme in the thiaminbiosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from1-deoxy-D-xylulose-5-phosphate, cysteine, and tyrosine. The structure of ThiK in the rhombohedral crystalform has been determined to 1.5 Å resolution and refined to a final R-factor of 21.6% (R-free 25.1%).The structures of the enzyme/Thz complex and the enzyme/Thz-phosphate/ATP complex have also beendetermined. ThiK is a trimer of identical subunits. Each subunit contains a large nine-stranded central-sheet flanked by helices. The overall fold is similar to that of ribokinase and adenosine kinase, althoughsequence similarity is not immediately apparent. The area of greatest similarity occurs in the ATP-bindingsite where several key residues are highly conserved. Unlike adenosine kinase and ribokinase, in whichthe active site is located between two domains within a single subunit, the ThiK active site it formed atthe interface between two subunits within the trimer. The structure of the enzyme/ATP/Thz-phosphatecomplex suggests that phosphate transfer occurs by an inline mechanism. Although this mechanism issimilar to that proposed for both ribokinase and adenosine kinase, ThiK lacks an absolutely conservedAsp thought to be important for catalysis in the other two enzymes. Instead, ThiK has a conserved cysteine(Cys198) in this position. When this Cys is mutated to Asp, the enzymatic activity increases 10-fold.Further sequence analysis suggests that another thiamin biosynthetic enzyme (ThiD), which catalyzes theformation of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by two sequential phosphorylation reactions, belongs to the same family of small molecule kinases.