Metabolic Flux in Both the Purine Mononucleotide and Histidine Biosynthetic Pathways Can Influence Synthesis of the Hydroxymethyl Pyrimidine Moiety of Thiamine in Salmonella enterica

Abstract

Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.Cellular metabolism requires the control of flux through a complex network of pathways. This control is accomplished by regulation at several levels. Regulation of gene transcription or mRNA translation can control individual pathways. Allosteric control of the enzyme catalyzing the first committed step of a pathway is another mechanism used by cells to regulate biosynthetic pathways. Regulatory effects resulting from interactions between pathways are less well understood, despite the fact that many metabolites are present in multiple pathways in the cell.The expanding network of defined pathways that affect the biosynthesis of thiamine provides an attractive model system to investigate metabolic integration. As depicted in Fig. 1, the biosynthetic pathway for the hydroxymethylpyrimidine (HMP) moiety of thiamine pyrophosphate (TPP) branches off from that for purine mononucleotides at the metabolite aminoimidazole ribotide (AIR). Relevant to the work presented here is a subsequent purine intermediate, aminoimidazole carboxamide ribotide (AICAR), that is also a by-product of the biosynthesis of histidine.The distribution of AIR at the purine-HMP branch point must account for the different level of purine mononucleotides and TPP required for growth (purines/TPP ratio, 1,000:1, based on auxotrophic requirements). The distribution of AIR between these pathways could be accomplished by kinetic parameters of the relevant enzymes; however, the first dedicated HMP biosynthetic enzyme is poorly understood, thus hampering direct tests of this possibility.While thiamine and purine mononucleotides are synthesized by pathways diverging at the metabolite AIR, the cellular pool of AICAR arises from both purine and histidine biosynthesis and is used exclusively for purine mononucleotide synthesis (28, 29). Since AICAR enters purine mononucleotide synthesis after the purine-HMP branch point, no interaction between AICAR and HMP synthesis was anticipated. Hence, it was surprising to find that mutants blocked in the utilization of AICAR (purH mutants) required both purines and thiamine to grow (16, 58). The hypothesis proposed to explain this requirement posited that inactivation of purH would result in the acc...

Plasticity in the Purine–Thiamine Metabolic Network of Salmonella

“…Methionine and lysine lower the cellular thiamine requirement of S. enterica by sparing succinyl-CoA (Enos-Berlage and Downs 1997). Additionally, although AICAR was required for the synthesis of AIR here, it also inhibited the ThiCcatalyzed reaction in vivo (Allen et al 2002). One possibility that is consistent with our data, as well as with Cells were grown in glucose medium with adenine and the indicated additions.…”

“…data from previous physiology studies on purH mutants, is that the inhibitory effect of AICAR on ThiC is antagonized by the presence of methionine and lysine in the medium (Westby and Gots 1969;Allen et al 2002). Taken together, these points suggest that multiple antagonistic effects combined in vivo to generate the growth properties that we detected.…”

“…Unless otherwise stated, cell density was measured as absorbance at 650 nm, and growth was routinely reported as specific growth rates [m ¼ ln(X/X 0 )/T] or the final cell density (OD 650 ) reached after 12-24 hr incubation at 37°with shaking (Downs 1992). When nutritional requirements were measured on solid medium, soft agar overlays were used as has been described (Allen et al 2002).…”

“…1 5-Amino-4-imidazole carboxamide ribotide (AICAR) is a metabolite that integrates the purine and histidine biosynthetic pathways, and it is a by-product metabolite formed in histidine biosynthesis that is an intermediate in purine biosynthesis (Figure 1) (Magasanik and Karibian 1960;Moyed and Magasanik 1960;Allen et al 2002). The last two reactions of purine biosynthesis convert AICAR to inosine monophosphate (IMP) and are catalyzed by phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (PurH, EC 2.1.2.3/3.5.4.10).…”

Anthranilate Synthase Can Generate Sufficient Phosphoribosyl Amine for Thiamine Synthesis in Salmonella enterica

“…The structure and function of networks created by the integration and overlap of metabolic processes are largely uncharacterized. The thiamine biosynthetic pathway in Salmonella enterica has direct biochemical connections with other metabolic pathways, including purine and isoprenoid biosynthesis (5,33,40), and indirect connections with a number of other cellular processes (1,16,19,25,36). Thus, this biosynthetic pathway serves as a focal point for studies to identify and characterize metabolic interactions, redundancy, and regulation that are essential in a robust metabolism.…”