Abstract
The enzyme fructose-1,6-bisphosphatase (FBP) is key regulatory point in gluconeogenesis. Mutants of Salmonella enterica lacking purH accumulate 5-amino-4-imidazole carboxamide ribotide (AICAR) and are unable to utilize glycerol as sole carbon and energy sources. The work described here demonstrates this lack of growth is due to inhibition of FBP by AICAR. Mutant alleles of fbp that restore growth on glycerol encode proteins resistant to inhibition by AICAR and the allosteric regulator AMP. This is the first report of biochemical characterization of substitutions causing AMP resistance in a bacterial FBP. Inhibition of FBP activity by AICAR occurs at physiologically relevant concentrations and may represent a form of regulation of gluconeogenic flux in Salmonella enterica.
Highlights
amino-4-imidazole carboxamide ribotide (AICAR) is a purine nucleotide biosynthetic intermediate and the substrate for AICAR transformylase/IMP cyclohydrolase, encoded by the purH gene in Salmonella enterica
AICAR is generated as a byproduct of histidine biosynthesis, released by the enzyme imidazole glycerol-phosphate synthase (Fig. 1A)
Bochner and Ames [1] proposed that 5amino-4-imidazole carboxamide riboside 5Ј-triphosphate formed from the accumulation of AICAR is an alarmone for C1-folate deficiency in S. enterica, this conclusion, in its simplest form, was not supported by subsequent work [2]
Summary
AICAR is a purine nucleotide biosynthetic intermediate and the substrate for AICAR transformylase/IMP cyclohydrolase, encoded by the purH gene in Salmonella enterica. 1 Supported by Biotechnology Traineeship Grant T32 GM08349 from the National Institutes of Health, a Louis and Elsa Thomsen Wisconsin Distinguished Fellowship Award, and the William H. 3 The abbreviations used are: AICAR, 5-amino-4-imidazole carboxamide ribotide; FBP, fructose-1,6-bisphosphatase; Fru-1,6-P, fructose 1,6-bisphosphate; Fru2,6-P, fructose 2,6-bisphosphate. AICAR has been shown to activate AMP-activated protein kinase [6], which plays a critical role in energy balance and the response to metabolic stress [7]. FBP is important for the regulation of flux between glycolysis and gluconeogenesis (Fig. 1B); as the enzyme is inhibited by AMP and fructose 2,6bisphosphate (Fru-2,6-P) [13]. E. coli FBP has been shown to be activated by Mg2ϩ and inhibited by AMP and Fru-2,6-P, similar to the mammalian enzymes, Mg2ϩ activation and AMP inhibition did not display cooperativity, as in the mammalian enzymes [20]. The crystal structure of E. coli FBP was reported; 33892 JOURNAL OF BIOLOGICAL CHEMISTRY
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