Mutational analysis of ThiH, a member of the radical S-adenosylmethionine (AdoMet) protein superfamily.
Thiamine pyrophosphate (TPP) is an essential cofactor for all forms of life. In Salmonella enterica, the thiH gene product is required for the synthesis of the 4-methyl-5-beta hydroxyethyl-thiazole monophosphate moiety of TPP. ThiH is a member of the radical S-adenosylmethionine (AdoMet) superfamily of proteins that is characterized by the presence of oxygen labile [Fe-S] clusters. Lack of an in vitro activity assay for ThiH has hampered the analysis of this interesting enzyme. We circumvented this problem by using an in vivo activity assay for ThiH. Random and directed mutagenesis of the thiH gene was performed. Analysis of auxotrophic thiH mutants defined two classes, those that required thiazole to make TPP (null mutants) and those with thiamine auxotrophy that was corrected by either L-tyrosine or thiazole (ThiH* mutants). Increased levels of AdoMet also corrected the thiamine requirement of members of the latter class. Residues required for in vivo function were identified and are discussed in the context of structures available for AdoMet enzymes.
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53726-4087, USA.
Norma C
NC
Martinez-Gomez
Matt
M
Robers
Diana M
DM
Downs
D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D029968:Escherichia coli Proteins / Q000235:genetics*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012436:S-Adenosylmethionine / Q000378:metabolism*; D019779:Salmonella enterica / Q000201:enzymology / Q000378:metabolism*; D013834:Thiamine Pyrophosphatase / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C486458:ThiH protein, E coli; D013844:Thiazoles; D014443:Tyrosine; D012436:S-Adenosylmethionine; D013834:Thiamine Pyrophosphatase; D010100:Oxygen