Comparative Genomic and Genetic Evidence on a Role for the OarX Protein in Thiamin Salvage

Abstract

Salvage pathways for thiamin and its thiazole and pyrimidine moieties are poorly characterized compared to synthesis pathways. A candidate salvage gene is oarX, which encodes a short-chain dehydrogenase/reductase. In diverse bacteria, oarX clusters on the chromosome with genes of thiamin synthesis, salvage, or transport and is preceded by a thiamin pyrophosphate riboswitch. Thiamin and its moieties can undergo oxidations that convert a side-chain hydroxymethyl group to a carboxyl group, or the thiazole ring to a thiazolone, causing a loss of biological activity. To test if OarX participates in salvage of the carboxyl or thiazolone products, we used a genetic approach in Corynebacterium glutamicum ATCC 14067, which is auxotrophic for thiamin's pyrimidine moiety. This strain could not utilize the pyrimidine carboxyl derivative. This excluded a role in salvaging this product and narrowed the function search to metabolism of the carboxyl or thiazolone derivatives of thiamin or its thiazole moiety. However, a ΔthiG (thiazole auxotroph) strain was not rescued by any of these derivatives. Nor did deleting oarX affect rescue by the physiological pyrimidine and thiazole precursors of thiamin. These findings reinforce the genomic evidence that OarX has a function in thiamin metabolism and rule out five logical possibilities for what this function is.

Figure 1

Canonical bacterial thiamin synthesis and salvage pathways. Enzymes: ThiD, hydroxymethylpyrimidine/phosphomethylpyrimidine kinase; ThiE, thiamin phosphate synthase; ThiG, thiazole synthase; ThiL, thiamin phosphate kinase; ThiN, thiamin pyrophosphokinase; ThiO, glycine oxidase; ThMPase, thiamin monophosphatase. Dehydroglycine can also be derived from tyrosine via ThiH. Compounds: AIR, 5-aminoimidazole ribonucleotide; HET, 5-(2-hydroxy-ethyl)-4-methylthiazole; HMP, 4-amino-5-hydroxymethyl-2-methylpyrimidine; HMP-P, HMP monophosphate; HMP-PP, HMP pyrophosphate; MTA, 4-methyl-5-thiazoleacetic acid; oxo-HET, oxo derivative of HET; oxo-thiamin, oxo derivative of thiamin; PCA, 2-methyl-4-amino-5-pyrimidinecarboxylic acid; ThiS-COSH, ThiS thiocarboxylate; HET-P, HET-phosphate. Damage products of thiamin and its thiazole or pyrimidine precursors are shown in blue and tied to the corresponding physiological compound with dashed blue lines.

Figure 2

Examples of chromosomal clustering of genes encoding OarX, FmnO, and acyl-CoA synthetase (ACS) with a thiamin pyrophosphate riboswitch (R) and thiamin synthesis, salvage, and transport genes. ThiD, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase; ThiE, thiamin phosphate synthase; ThiF, ThiS adenylyltransferase; ThiG, thiazole synthase; ThiM, HET kinase; ThiO, glycine oxidase; ThiS, sulfur carrier protein; ThiV, predicted thiamin precursor transporter; YkoCDE, ECF thiamin transporter components; TenA, thiamin pyrimidine moiety salvage enzyme; TenI, thiazole tautomerase; and Tbp, thiamin-binding protein.

Figure 3

Growth responses of the C. glutamicum ATCC 14067 wild-type strain or its ΔthiG mutant to oxidative degradation products of thiamin. Growth was measured after 24 h. The corresponding physiological precursor (HMP or HET) and thiamin served as benchmarks. (a) Response of the wild-type strain to PCA. (b) Response of the ΔthiG strain to MTA. (c) Response of the ΔthiG strain to oxo-HET. (d) Response of the ΔthiG strain to oxo-thiamin. (e) Response of the ΔthiG strain to thiamin acetic acid. Data are from three independent cultures. Mean values are shown by horizontal bars (±standard error of the mean).