Steve Ealick's Research Group


Thiaminase-I from Bacillus thiaminolyticus


PDB files:

2THI (Form I, chains A and B)

3THI (Form II, chain A)

4THI (Form II, with bound inhibitor: 4-amino-6-chloro-2,5-dimethylpyrimidine)

Description:

Two thiaminases, which catalyze the degredation of thiamin (vitamin B1) have been isolated. Thiaminase-I catalyzes a reaction in which thiazole is replaced by a variety of organic nucleophiles, while thiaminase-II uses water as the nucleophile. In this paper, we determined the structure of thiaminase-I in two crystal forms (I and II) as well as the structure of form II with an inhibitor. In addition, we proposed a double addition-elimination reaction as the mechanism of action.

Thi-I is a monomer in its biological active form, with two distinct globular domains (the N-domain and the C domain). This structure was also determined at 2.0 Å resolution by molecular replacement. This image shows Form II of thiaminase-I with the inhibitor 4-amino-2,5-dimethylpyrimidine covalently bonded to C113. This analogue, which functions as a mechanism-based inactivating agent, confirms the location of the active site.

 

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The active site lies in a cleaft between the N-domain and the C-domain. The structure clearly shows C113 bonding to the pyrimidine. E241 is a possible active site base involved in deprotonation of Cys113, as evidenced by the inactivity of the E241G mutant.

Click the image to enlarge.

 

References:

Campobasso N, Costello CA, Kinsland C, Begley TP, and Ealick SE. Crystal Structure of Thiaminase-I from Bacillus thiaminolyticus at 2.0 Å Resolution. Biochemistry 37:15981-15989 (1998).

Campobasso N., Begun J., Costello CA, Begley TP and Ealick SE. Crystallization and Preliminary X-ray Analysis of Thiaminase I from Bacillus thiaminolyticus: Space Group Change Upon Freeing of Crystals. Acta Crystallogr. D 54:448-450 (1998).



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