Mutants of Salmonella typhimurium LT-2 deficient in nicotinamidase activity (pncA) or nicotinic acid phosphoribosyltransferase activity (pncB) were isolated as resistant to analogs of nicotinic acid and nicotinamide. Information obtained from interrupted mating experiments placed the pncA gene at 27 units and the pncB gene at 25 units on the S. typhimurium LT-2 linkage map. A major difference in the location of the pncA gene was found between the S. typhimurium and Escherichia coli linkage maps. The pncA gene is located in a region in which there is a major inversion of the gene order in S. typhimurium as compared to that in E. coli. Growth experiments using double mutants blocked in the de novo pathway to nicotinamide adenine dinucleotide (NAD) (nadc) and in the pyridine nucleotide cycle (pnc) at either the pncA or pncB locus, or both, have provided evidence for the existence of an alternate recycling pathway in this organism. Mutants lacking this alternate cycle, pncC, have been isolated and mapped via cotransduction at 0 units. Utilization of exogenous NAD was examined through J. S. GOt& SA797 purC proA46 ilv-K. E. Sandersonb 405 rha-461 str' M-10 fla-56 fim HU180 metP760gal J. S. Gots trpA49 trpA49 K. E. Sanderson Hfr SA534 137 cwc serA13 rfa-3058
Three catalytic domains of the Escherichia coli carbamoyl-phosphate synthetase (EC 6.3.5.5) have been identified in previous studies. These include the glutamine amide-N transfer domain in the carboxyl-terminal half of the glutaminase component and at least two adenine nucleotide binding sites in the synthetase component. To delineate the domains involved in subunit interactions, we have examined the effects of deletions and point mutations in the glutaminase and synthetase subunits on formation of the af6 holoenzyme. Deletion of the amino-terminal third of the glutaminase subunit abolishes interactions with the synthetase subunit, suggesting that this domain functions to stabilize the complex. Two subunit binding domains have been identified in the synthetase subunit. They are homologous to one another and are located in the amino-terminal and central regions of the synthetase component. These domains are adjacent to regions of the synthetase previously proposed to be involved in ATP binding and, possibly, activation of CO2. The new data enlarge the definition of the structural and functional domains in the two interdependent components of carbamoyl-phosphate synthetase.In Escherichia coli and most other bacteria, carbamoyl phosphate, an intermediate of arginine and pyrimidine biosynthesis, is synthesized by a single enzyme (1), glutaminedependent carbamoyl-phosphate synthetase [carbon dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]. Bacterial carbamoyl-phosphate synthetase is composed of two subunits (2, 3). The smaller subunit, encoded by carA (4), cleaves glutamine and transfers the resultant NH3 to the larger synthetase subunit for carbamoyl phosphate synthesis (5). The glutaminase subunit of carbamoyl-phosphate synthetase appears to comprise two evolutionarily distinct domains (6). The carboxyl-terminal half is homologous to a large number of glutamine amidotransferases (6-8) and is most likely concerned with the transfer of glutamine amide N to the synthetase subunit. The function of the amino-terminal half of the glutaminase subunit is still conjectural. The structure of the large synthetase subunit, encoded by carB (9), is also interesting. This protein is composed oftwo homologous halves. Each half has been proposed to contain at least one composite or, possibly, two separate adenine nucleotide binding sites (9, 10) whose precise functions in catalysis and regulation are not known.As part of a broader study aimed at delineating the structural and functional domains of the glutaminase and synthetase subunits, we have undertaken a mutational analysis of the E. coli carbamoyl-phosphate synthetase. Different regions of each subunit have been deleted, and the mutant proteins have been analyzed for their abilities to form a physical complex. The results of mutational analysis have allowed us to demarcate the regions of the glutaminase and synthetase subunits critical for subunit interactions and for the expression of their respective catalytic functions.
The roles played by the N-linked glycans of the Friend murine leukemia virus envelope proteins were investigated by site-specific mutagenesis. The surface protein gp70 has eight potential attachment sites for N-linked glycan; each signal asparagine was converted to aspartate, and mutant viruses were tested for the ability to grow in NIH 3T3 fibroblasts. Seven of the mutations did not affect virus infectivity, whereas mutation of the fourth glycosylation signal from the amino terminus (gs4) resulted in a noninfectious phenotype. Characterization of mutant gene products by radioimmunoprecipitation confirmed that glycosylation occurs at all eight consensus signals in gp70 and that gs2 carries an endoglycosidase H-sensitive glycan. Elimination of gs2 did not cause retention of an endoglycosidase H-sensitive glycan at a different site, demonstrating that this structure does not play an essential role in envelope protein function. The gs3- mutation affected a second posttranslational modification of unknown type, which was manifested as production of gp70 that remained smaller than wild-type gp70 after removal of all N-linked glycans by peptide N-glycosidase F. The gs4- mutation decreased processing of gPr80 to gPr90, completely inhibited proteolytic processing of gPr90 to gp70 and Pr15(E), and prevented incorporation of envelope products into virus particles. Brefeldin A-induced mixing of the endoplasmic reticulum and parts of the Golgi apparatus allowed proteolytic processing of wild-type gPr90 to occur in the absence of protein transport, but it did not overcome the cleavage defect of the gs4- precursor, indicating that gs4- gPr90 is resistant to the processing protease. The work reported here demonstrates that the gs4 region is important for env precursor processing and suggests that gs4 may be a critical target in the disruption of murine leukemia virus env product processing by inhibitors of N-linked glycosylation.
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.
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