Action of the Thiamine Antagonist Bacimethrin on Thiamine Biosynthesis

Abstract

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thi operon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the thiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.Thiamine is synthesized de novo by many bacteria. Although the general outline of the thiamine biosynthetic pathway in enteric microorganisms is known, the individual reactions in this pathway are not yet fully understood. Thiamine pyrophosphate (TPP), the biologically active cofactor, is generated by the condensation of two independently synthesized intermediates, 4-methyl-5-(␤-hydroxyethyl)thiazole (THZ) phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) pyrophosphate. In vivo labeling studies demonstrated that the carbon atoms in HMP were derived from the purine intermediate aminoimidazole ribonucleotide (AIR), although the biochemical details of this conversion have not been elucidated (2). Mutations causing an unconditional requirement for exogenous HMP or thiamine map to a single gene, thiC. In vivo labeling also determined the metabolic precursors to the THZ moiety, and several genes have been implicated in this synthesis (2, 18). Our current understanding of the synthesis and salvage of TPP is presented in Fig. 1.While the low cellular requirement for thiamine has complicated the detailed analysis of its biosynthetic pathway, this characteristic has made thiamine synthesis an attractive model system for dissecting the integration of metabolic pathways (25). Characterization of mutations that perturbed, but did not eliminate, thiamine synthesis has resulted in the identification of a number of loci required for optimal synthesis of this vitamin and has increased our understanding of the integration of thiamine biosynthesis with central cellular processes (1, 5, 6, 8, 10-12, 24b, 25, 27).Use of metabolic inhibitors, or analogs, has provided insight in the studies of a number of metabolic pathways, including histidine (17), folate (3), and branched-chain amino acids (19)(20)(21). Bacimethrin is a thiamine antagonist produced by grampositive bacteria, including Streptomyces albus (7) and Bacillus megaterium (29) (Fig. 1). Previous reports showed that the inhibitory effect of bacimethrin on defined medium could be overcome by exogenous addition of thiamine or the HMP moiety (7).Wild-type Salmonella enterica serovar Typhimurium strain LT2 was unable to grow on solid minimal medium in the presence of 130 nM bacimethrin, and resistant mutants arose spontaneously at a frequency of ϳ10 Ϫ7 (data not shown). Growth of the wild-type strain was restored ...

Pre- and post-weaning diet alters the faecal metagenome in the cat with differences in vitamin and carbohydrate metabolism gene abundances

“…While it has been suggested that kibbled diets may increase thiamine requirements due to their high carbohydrate content14, recent literature suggests that thiamine concentrations in canned food may be below the recommended amounts for adult cats15. As for all mammals, thiamine cannot be synthesised by the cat, instead it relies on microbial de novo biosynthesis54 or ‘salvaging’ pathways545556.…”

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

“…3 The toxicity of the bacimethrin on expression of thiamin biosynthetic genes was reported to be weak in comparison with the effect of thiamin itself. 2,5 An alternative explanation of bacimethrin toxicity was that upon conversion to MeOThDP, it could inhibit some or all of the ThDP-dependent enzymes in the cell. In experiments with Escherichia coli cells growing on minimal medium with bacimethrin and nutritional supplements, the OGDHc-ec, 1-deoxy- d -xylulose 5-phosphate synthase (DXPS), and transketolase were identified as major targets for inhibition by MeOThDP.…”

A Genetic Screen for the Identification of Thiamin Metabolic Genes

“…A recent study demonstrated that the thiamin biosynthetic enzymes convert bacimethrin to 2Ј-methoxythiamin pyrophosphate (MeO-TPP), which inhibits thiamin-utilizing enzymes (13). In the presence of low levels of exogenous thiamin, cell growth is not inhibited by bacimethrin (14,15). CF 3 -HMP is converted by HMP-P kinase (ThiD) to CF 3 -HMP pyrophosphate (CF 3 -HMP-PP), which inhibits thiamin phosphate synthase (ThiE) (16).…”