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

Abstract

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.Thiamine pyrophosphate is an essential cofactor that stabilizes acyl carbanions generated by several enzymes in carbohydrate metabolism such as transketolase, ␣-ketoacid decarboxylase, ␣-ketoacid dehydrogenase, and acetolactate synthase. The biosynthesis of thiamine pyrophosphate involves the separate formation of the 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) 1 and thiazole monophosphate (THZ-P) moieties (Fig. 1). These moieties are then coupled and phosphorylated, forming thiamine pyrophosphate (1). In Escherichia coli and Salmonella at least seven genes, thiFSGH, thiI, iscS, and dxs, are required to convert 1-deoxy-D-xylulose phosphate, L-cysteine and L-tyrosine to THZ-P (2-4). Many of the mechanistic aspects of this conversion have been worked out in vitro (5-8). Our understanding of thiazole biosynthesis has increased dramatically with the reconstitution of the pathway in cell-free extracts by two groups (9, 10). Although these groups used proteins from Bacillus subtilis and E. coli, the proposed mechanisms are similar. The primary difference between these pathways is the generation of the glycine imine intermediate from which the C-2 and N-3 of the thiazole product are derived. In B. subtilis, ThiO catalyzes the oxidation of glycine to form the imine (11), whereas in E. coli, the imine intermediate is proposed to be the product of ThiH (10). In the case of ThiH, it is suggested that an interaction between S-adenosylmethionine (AdoMet), and the [Fe-S] cluster allows the formation of the 5Ј-deoxyadenosyl radical, generating a tryosyl radical that results in the glycine imine via a series of steps (10).Genetic studies identified a connection between thiamine biosynthesis and [Fe-S] cluster metabolism in Salmonella enterica and suggested that ThiH was the weak link in thiazole synthesis (12). ThiH is a 45-kDa protein that was identified as a member of the radical AdoMet superfamily of proteins (13). Proteins in this superfamily are characte...

Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

“…Complementation of two of the growth defects required restoration of the low flux thiamine biosynthesis pathway (the amount of thiamine pyrophosphate in wild-type S. enterica grown in minimal glucose medium is 41.4 pmol/mg dry weight (46)). Stains lacking apbC and yggX (or purF) are thiamine auxotrophs likely because of poor cluster occupancy of ThiH/ThiC (47,48,80). Complementation of a third growth defect required function of the high flux tricarballylate catabolic metabolic pathway (35,37,49,50).…”

Thiazole Synthase from Escherichia coli

“…These observations suggest the possibility of chemical iron sulfur cluster reconstitution and an AdoMet-driven reaction, most likely that of ThiH. Mutagenesis of the highly conserved cysteine-rich motif from Salmonella enterica ThiH suggested that these residues function as ligands to the [4Fe-4S] cluster (16).…”

“…Whole cell feeding studies in E. coli and knock-out mutants thereof indicated that Thz-P synthesis is dependent on the precursors tyrosine, cysteine, and 1-deoxy-xylulose-5-phosphate (Dxp) and the enzymes ThiFSGH, ThiI, and IscS (4,10,(12)(13)(14)(15)(16). The Thz-P pathway in B. subtilis is significantly different, since the oxygensensitive iron sulfur cluster-containing enzyme ThiH is replaced by ThiO, an oxygen-tolerant FAD-dependent oxidase.…”

Lack of YggX Results in Chronic Oxidative Stress and Uncovers Subtle Defects in Fe-S Cluster Metabolism in Salmonella enterica

“…Along with mutations in the isc operon, mutations in the four loci described above cause a conditional thiamine auxotrophy in S. enterica attributed to impaired assembly or repair of the Fe-S cluster in the ThiH enzyme (14,25,42). In addition to this thiamine requirement, one of the defining features of this class of mutants was the observation that the expression of yggX suppressed the thiamine auxotrophy of each (13).…”

“…In S. enterica, rseC is the fourth gene in the rpoE operon, although no connection between rseC and rpoE regulation has been found. In E. coli, mutations in this gene were isolated because they caused constitutive expression of the SoxRS regulon (18), leading to the hypothesis that RseC is involved in the reduction of the Fe-S clusters in SoxR after recovery from oxidative stress (18).Along with mutations in the isc operon, mutations in the four loci described above cause a conditional thiamine auxotrophy in S. enterica attributed to impaired assembly or repair of the Fe-S cluster in the ThiH enzyme (14,25,42). In addition to this thiamine requirement, one of the defining features of this class of mutants was the observation that the expression of yggX suppressed the thiamine auxotrophy of each (13).…”