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Pyridoxal 5′-phosphate (PLP) is an essential cofactor for organisms in all three domains of life. Despite the central role of PLP, many aspects of vitamin B(6) metabolism, including its integration with other biological pathways, are not fully understood. In this study, we examined the metabolic perturbations caused by the vitamin B(6) antagonist 4-deoxypyridoxine (dPN) in a ptsJ mutant of Salmonella enterica serovar Typhimurium LT2. Our data suggest that PdxK (pyridoxal/pyridoxine/pyridoxamine kinase [EC 2.7.1.35]) phosphorylates dPN to 4-deoxypyridoxine 5′-phosphate (dPNP), which in turn can compromise the de novo biosynthesis of PLP. The data are consistent with the hypothesis that accumulated dPNP inhibits GlyA (serine hydroxymethyltransferase [EC 2.1.2.1]) and/or GcvP (glycine decarboxylase [EC 1.4.4.2]), two PLP-dependent enzymes involved in the generation of one-carbon units. Our data suggest that this inhibition leads to reduced flux to coenzyme A (CoA) precursors and subsequently decreased synthesis of CoA and thiamine. This study uncovers a link between vitamin B(6) metabolism and the biosynthesis of CoA and thiamine, highlighting the integration of biochemical pathways in microbes. IMPORTANCE PLP is a ubiquitous cofactor required by enzymes in diverse metabolic networks. The data presented here expand our understanding of the toxic effects of dPN, a vitamin B(6) antagonist that is often used to mimic vitamin B(6) deficiency and to study PLP-dependent enzyme kinetics. In addition to de novo PLP biosynthesis, we define a metabolic connection between vitamin B(6) metabolism and synthesis of thiamine and CoA. This work provides a foundation for the use of dPN to study vitamin B(6) metabolism in other organisms.

YggS (COG0325) is a pyridoxal 5′-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B6 vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli. Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5′-phosphate (PNP). Data herein show that strains lacking YggS accumulated ∼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5′-phosphate (PMP) oxidase credited with interconverting B6 vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae. The impact of a yggS deletion on the intracellular and extracellular levels of B6 vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence. IMPORTANCE Pyridoxal 5′-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B6 vitamer content can be detrimental, notably causing B6-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B6 vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.

YggS (COG0325) is a pyridoxal 5′-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B(6) vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli. Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5′-phosphate (PNP). Data herein show that strains lacking YggS accumulated ∼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5′-phosphate (PMP) oxidase credited with interconverting B(6) vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae. The impact of a yggS deletion on the intracellular and extracellular levels of B(6) vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence. IMPORTANCE Pyridoxal 5′-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B(6) vitamer content can be detrimental, notably causing B(6)-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B(6) vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.

Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain ( mxaF xoxF1 xoxF2 ; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF , was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La 3+ ). ExaF was purified from cells grown with both calcium (Ca 2+ ) and La 3+ and with Ca 2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La 3+ over Ca 2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca 2+ -plus-La 3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca 2+ -only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La 3+ is present, expanding the role of lanthanides to multicarbon metabolism. IMPORTANCE ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 μM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. IMPORTANCE The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements.