Structure of trifunctional THI20 from yeast.

In a recently characterized thiamin-salvage pathway, thiamin-degradation products are hydrolyzed by thiaminase II, yielding 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). This compound is an intermediate in thiamin biosynthesis that, once phosphorylated by an HMP kinase, can be used to synthesize thiamin monophosphate. Here, the crystal structure of Saccharomyces cerevisiae THI20, a trifunctional enzyme containing an N-terminal HMP kinase/HMP-P kinase (ThiD-like) domain and a C-terminal thiaminase II (TenA-like) domain, is presented. Comparison to structures of the monofunctional enzymes reveals that while the ThiD-like dimer observed in THI20 resembles other ThiD structures, the TenA-like domain, which is tetrameric in all previously reported structures, forms a dimer. Similarly, the active site of the ThiD-like domain of THI20 is highly similar to other known ThiD enzymes, while the TenA-like active site shows unique features compared with previously structurally characterized TenAs. In addition, a survey of known TenA structures revealed two structural classes, both of which have distinct conserved features. The TenA domain of THI20 possesses some features of both classes, consistent with its ability to hydrolyze both thiamin and the thiamin-degradation product 2-methyl-4-amino-5-aminomethylpyrimidine.

[1]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[2]  A. Chatterjee,et al.  Domain organization in Candida glabrata THI6, a bifunctional enzyme required for thiamin biosynthesis in eukaryotes. , 2010, Biochemistry.

[3]  L. Cendron,et al.  Structural and mutational analysis of TenA protein (HP1287) from the Helicobacter pylori thiamin salvage pathway – evidence of a different substrate specificity , 2009, The FEBS journal.

[4]  T. Begley,et al.  The structural and biochemical foundations of thiamin biosynthesis. , 2009, Annual review of biochemistry.

[5]  K. Akaji,et al.  Involvement of thiaminase II encoded by the THI20 gene in thiamin salvage of Saccharomyces cerevisiae. , 2008, FEMS yeast research.

[6]  T. Begley,et al.  A new thiamin salvage pathway. , 2007, Nature chemical biology.

[7]  K. Nosaka.  Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae , 2006, Applied Microbiology and Biotechnology.

[8]  T. Begley,et al.  Thi20, a remarkable enzyme from Saccharomyces cerevisiae with dual thiamin biosynthetic and degradation activities. , 2005, Bioorganic chemistry.

[9]  B. Rost,et al.  The 2.35 A structure of the TenA homolog from Pyrococcus furiosus supports an enzymatic function in thiamine metabolism. , 2005, Acta crystallographica. Section D, Biological crystallography.

[10]  T. Begley,et al.  Structural characterization of the regulatory proteins TenA and TenI from Bacillus subtilis and identification of TenA as a thiaminase II. , 2005, Biochemistry.

[11]  T. Mizote,et al.  Biosynthesis of hydroxymethylpyrimidine pyrophosphate in Saccharomyces cerevisiae , 2005, Current Genetics.

[12]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[13]  Tadhg P Begley,et al.  Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 A resolution. , 2002, Structure.

[14]  I. Mathews,et al.  Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 A resolution. , 2000, Biochemistry.

[15]  B. Dujon,et al.  Genetic redundancy and gene fusion in the genome of the baker's yeast Saccharomyces cerevisiae : functional characterization of a three‐member gene family involved in the thiamine biosynthetic pathway , 1999, Molecular microbiology.

[16]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[17]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[18]  A. Pang,et al.  Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis , 1991, Journal of bacteriology.