Isolation of NAD cycle mutants defective in nicotinamide mononucleotide deamidase in Salmonella typhimurium.
The NAD or pyridine nucleotide cycle is the sequence of reactions involved in the breakdown of NAD to nicotinamide mononucleotide (NMN) and regeneration of NAD. This cycle is fivefold more active during aerobic growth of Salmonella typhimurium and under this condition breaks down half of the NAD pool every 90 min. DNA ligase is known to convert NAD to NMN but is only a minor contributor to the NAD cycle during aerobic growth. The dominant aerobic route of NMN formation is otherwise uncharacterized. Accumulated NMN generated by either of these routes is potentially dangerous in that it can inhibit the essential enzyme DNA ligase. The reactions which recycle NMN to NAD may serve to minimize the inhibition of ligase and other enzymes by accumulated NMN. The predominant recycling reaction in S. typhimurium appears to be NMN deamidase, which converts NMN directly to the biosynthetic intermediate nicotinic acid mononucleotide. Mutants defective in this recycling step were isolated and characterized. By starting with a ligase-deficient (lig mutant) parent strain that requires deamidase to assimilate exogenous NMN, two classes of mutants that are unable to grow on minimal NMN media were isolated. One class (pncC) maps at 83.7 min and shows only 2% of the wild-type levels of NMN deamidase. Under aerobic conditions, a lig+ allele allows a pncC mutant to grow on NMN and restores some deamidase activity. This growth ability and enzyme activity are not found in lig+ strains grown without oxygen. This suggests that the existence of a second NMN deamidase (pncL) dependent on ligase and stimulated during aerobic growth. The second class of mutants (pncD) gains a requirement for isoleucine plus valine with growth in the presence of exogenous NMN. We propose that pncD mutations reduce the activity of an ilv biosynthetic enzyme that is naturally sensitive to inhibition by NMN.
Cheng
W
W
Biology Department, University of Utah, Salt Lake City 84112, USA.
Roth
J
J
D000581:Amidohydrolases / Q000235:genetics*; D002874:Chromosome Mapping; D011088:DNA Ligases / Q000235:genetics; D008040:Genetic Linkage; D006358:Hot Temperature; D016254:Mutagenesis, Insertional; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D009537:Nicotinamide Mononucleotide / Q000378:metabolism*; D010641:Phenotype; D011489:Protein Denaturation; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D009243:NAD; D009537:Nicotinamide Mononucleotide; D000581:Amidohydrolases; C021861:nicotinamidenucleotide amidase; D011088:DNA Ligases