Steve Ealick's Research Group


4-Methyl-5-β-Hydroxyethylthiazole kinase from Bacillus subtilis


PDB files:

1C3Q (ThiM/Thz form II)

1EKQ (ThiM/Thz form I)

1EKK (ThiM)

1ESJ (ThiM(C198S))

1ESQ (ThiM(C198S)Thz-P/MgATP)

Description:

4-Methyl-5-β-hydroxyethylthiazole kinase (ThiM; previously referrred to as ThiK) catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-β-hydroxyethylthiazole (Thz). This enzyme is a salvage enzyme in the thiamin biosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from l-deoxy-o-xylulose-5-phosphate, cysteine, and tyrosine. The structure of ThiM in the rhombohedral crystal form has been determined to 1.5 Å resolution and refined to a final R-factor of 21.6% (R-free 25.1%). In addition, the structure of a monoclinic form with Thz and the structures of two mutants were determined.

The subunit contains a large nine-stranded central β-sheet flanked by helices. 52% of the amino acids are in α-helices and 14% are in β-sheets. The rest of the structure is composed of loops and coils. The monomer is structurally similar to adenosine kinase and ribokinase. When the subunits of the three enzymes are superimposed, the ATP-binding sites superimpose closely nd the residues of the β-sheet flaps correspond to residues in the adjacent subunit of ThiM.

 

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ThiM is a trimer of identical subunits. In rhombohedral crystals, the trimer axis is along the crystallographic three-fold axis. The overall structural features in the monoclinic crystal are identical to that in the rhombohedral crystal; however, the monoclinic crystal forms a complete trimer with the three-fold noncrystallographic axis approximately perpendicular to the crystallographic two-fold axis.

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The ThiM trimer contains three identical active sites. Unlike adenosine kinase and ribokinase, in which the active site is located between two domains within a single subunit, the ThiM active site is formed at the interface between two subunits within the trimer. The binding site of Thz-P, ATP, and Mg2+ is shown at the right. For a close-up of the residues at the active site, click here. Note that the residues from the two different subunits are color-code by bond color and that water molecules are shown as red spheres.

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Reference:

Campobasso N, Mathews II, Begley TP and Ealick SE. Crystal Structure of 4-Methyl-5-β-hydroxyethylthiazole Kinase from Bacillus subtilis at 1.5 Å Resolution. Biochemistry 39:7868-7877 (2000).



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