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J. Biol. Chem., Vol. 279, Issue 39, 40505-40510, September 24, 2004

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Mutational Analysis of ThiH, a Member of the Radical S-Adenosylmethionine (AdoMet) Protein Superfamily^*

Norma C. Martinez-Gomez, Matt Robers, and Diana M. Downs

From the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53726-4087

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- 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.

Received for publication, April 9, 2004 , and in revised form, July 1, 2004.

^* The competitive grant programs of the National Science Foundation funded this work through Grant MCB0096513. The J. S. McDonnell Foundation provided funds from a 21st Century Scientist Scholars Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 1710 University Ave., Madison, WI 53726-4087. Tel.: 608-265-4630; Fax: 608-265-7909; E-mail: downs{at}bact.wisc.edu.

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