Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide mononucleotide deamidase and characterization of pnuA mutants defective in nicotinamide mononucleotide transport

Abstract

The enzyme nicotinamide mononucleotide deamidase, an integral component of the proposed four-membered pyridine nucleotide cycle (PNC IV), has been demonstrated in extracts of Salmonella typhimurium LT2. The enzyme has an optimum pH of 8.7 and deamidates nicotinamide mononucleotide, forming nicotinic acid mononucleotide. Sigmoidal kinetic data suggest that this enzyme may be allosteric and therefore an important regulatory component of pyridine nucleotide cycle metabolism. Mutants previously designated pncC in anticipation of their lacking nicotinamide mononucleotide deamidase were examined and found to have normal levels of this enzyme. [14C]nicotinamide mononucleotide uptake studies, however, revealed a defect in the transport ofthis compound. Accordingly, the genetic designation for this locus was changed to pnuA to reflect its involvement in pyridine nucleotide uptake. Evidence is presented for the existence of two separate nicotinamide mononucleotide transport systems.

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities

“…Sal-moneUa typhinurium was shown to posses at least two PNCs: a four-membered cycle referred to as PNC IV, and a six-membered cycle referred to as PNC VI ( Fig. 1) (7,9,18). Although some of the enzymes in these cycles have not previously been demonstrated in vitro, both have been shown to recycle NAD in vivo, with PNC IV being the predominant cycle (6).…”

“…The present investigation was initiated to demonstrate the presence or absence of nicotinamide mononucleotide (NMN) glycohydrolase, NAD pyrophosphatase, and NAD glycohydrolase in cell-free extracts of S. typhimurium. NMN glycohydrolase and NAD pyrophosphatase, although suggested in S. typhimurium by several studies (7,9,18), have not been assayed previously in vitro. The results presented show that both of these enzymes exist and reveal the presence of two NAD glycohydrolase activities.…”

“…JF34 is an nadAl mutant of t*pA49 whose isolation and characterization were described elsewhere (7). The cultural conditions used for growth were described previously (18). Chemicals and reagents.…”

“…Nicotinic acid (NA), NAm, NMN, nicotinic acid mononucleotide (NAMN), desamido-NAD, NAD, NADH, NADP, and thionicotinamide adenine dinucleotide were purchased from Sigma Chemical Co., St. Louis, Mo. and were checked for purity by using chromatographic techniques described previously (7,17 zymatic degradation with snake venom phosphodiesterase I as described previously (18). All other chemicals were of analytical quality.…”

NAD metabolism in Vibrio cholerae.

“…Protein determinations were made as described by Lowry et al (15). Previous work from this laboratory with Salmonella typhimurium has provided evidence for three NAD recycling pathways (8,13). Therefore, crude extracts of V. cholerae 569B were assayed for several pyridine nucleotide cycle components at various pH values to determine which of these pathways may function.…”