Steve Ealick's Research Group


ThiO from B. subtilis


PDB file:

1NG3 (with acetyl glycine)

1NG4 (native)

Description:

In B. subtilisThiO is essential for the biosynthesis of the thiazole moiety of thiamin pyrophosphate. It is an FAD-dependent glycine oxidase.

ThiO is a flavoprotein that has two structural domains: a FAD binding domain and a substrate binding domain The FAD binding domain is made up of a six-stranded β-sheet. The sheet is flanked by a six α-helix bundle on one side and by a three-stranded β-sheet and another α-helix on the other side. The substrate binding domain consists of a mixed, eight-stranded β-sheet flanked by three α-helices. Bound FAD and N-acetylglycine are shown as stick models.


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This enzyme is a homotetramer in its active form with 222 point symmetry. This view is along the crystallographic 2-fold axis with the non-crystallographic 2-fold vertical. Each of the monomers of the tetramer makes contacts with the other three monomers. Bound FAD and N-acetylglycine are shown as stick models.


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FAD binds to ThiO in an extended conformation and forms 17 hydrogen bonds with the protein atoms (14 main chain, three side chain) and nine additional hydrogen bonds with water molecules. The N-acetylglycine is bound on the re side of the isoalloxazine ring. One carboxylate oxygen atom forms a hydrogen bond to the NE atom of Arg302 while the other forms a hydrogen bond with NH2 of Arg302 . The acetyl group is near the guanidinium group of Arg329. The positioning of the glycine places the Ca carbon atom about 3.5 Å from the N(5) atom of the isoalloxazine ring.

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Reference:

Settembre EC, Dorrestein PC, Park J-H, Augustine A, Begley TP and Ealick SE. Structural and Mechanistic Studies on ThiO, a Glycine Oxidase Essential for Thiamin Biosynthesis in Bacillus subtilis. Biochemistry 42:2971-2981 (2003).



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