Inhibition of Fructose-1,6-bisphosphatase by Aminoimidazole Carboxamide Ribotide Prevents Growth of Salmonella enterica purH Mutants on Glycerol

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.An efficient cellular metabolism that can adapt to changing environmental conditions requires the integration of many biochemical pathways. Subtle interactions that exist between pathways in the metabolic network are in general not well understood. One level of these interactions involves metabolites common to multiple pathways. Cellular metabolites can impact physiology through roles in transcriptional regulation, translational regulation, or by mediating allosteric effects on key enzymes. One such central metabolite with regulatory capabilities is 5-amino-4-imidazole carboxamide ribotide (AICAR).

Amino-4-Imidazolecarboxamide Ribotide Directly Inhibits Coenzyme A Biosynthesis in Salmonella enterica

“…DM2 (purH355) is a representative purH mutant strain with a previously described frameshift mutation that eliminates all PurH activity in the cell (6). DM2 and other purH mutants of S. enterica required exogenous addition of the HMP moiety of thiamine to grow in glucose medium with adenine (3)(4)(5).…”

“…Amino-4-imidazolecarboxamide ribotide (AICAR) is an intermediate in the de novo purine biosynthetic pathway and its accumulation impacts a variety of metabolic processes in a number of organisms such as Salmonella enterica (3)(4)(5)(6), Escherichia coli (8), Saccharomyces cerevisiae (9), and humans (11). AICAR is also generated as a by-product during histidine biosynthesis and recycled by subsequent entry into the purine biosynthetic pathway in organisms that synthesize histidine ( Fig.…”

“…Much of the work defining the consequences of AICAR accumulation has been in eukaryotic cell lines, where AICAR has been associated with disease treatments, such as methotrexate, and has shown therapeutic potential with regard to carbohydrate and lipid metabolism (15)(16)(17)(18)(19) and tumor cell proliferation (20,21). In bacteria, AICAR is linked to metabolic disturbances in thiamine biosynthesis (3)(4)(5), gluconeogenesis (6), and purine metabolism (8). In cases where it has been characterized, the mechanism of AICAR action is conserved between humans and bacteria, a finding that emphasizes the value of understanding the diverse targets of AICAR in a bacterial system.…”

In Salmonella enterica , 2-Methylcitrate Blocks Gluconeogenesis

“…These activators and inhibitors interconvert the FBPase homotetramer between its active R state and inactive T state (25,57). Specific allosteric inhibitors of FBPase include AMP (51), glucose 6-phosphate (26), and 5-amino-4-imidazole carboxamide ribotide (AICAR) (21). Activators include phosphoenolpyruvate (PEP), citrate, and sulfate ions (24,25).…”

Plasticity in the Purine–Thiamine Metabolic Network of Salmonella

“…In S. enterica, the thiamine requirement of a purH mutant (Yura 1956;Newell and Tucker 1968;Downs 1987) and the inability of the same mutant to grow on glycerol (Burton 1971;Dougherty et al 2006) have been attributed to the accumulation of AICAR. In Rhizobium etli, genetic studies showed that AICAR accumulation affected cellular processes such as the production of the cytochrome terminal oxidase cbb 3 (Soberon et al 1997) and the infection stages of symbiosis (Newman et al 1994).…”