Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro.
BACKGROUND
Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD+ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD+, especially for one of the alleles, nadD72.


RESULTS
In this study these two mutant proteins have been further characterized together with ten new mutant variants. Of the, in total, twelve mutations four are in a conserved motif in the C-terminus and eight are in the active site. We have tested the activity of the enzymes in vitro and their effect on the growth phenotype in vivo. There is a very good correlation between the two data sets.


CONCLUSIONS
The mutations in the C-terminus did not reveal any function for the conserved motif. On the other hand, our data has lead us to assign amino acid residues His-19, Arg-46 and Asp-109 to the active site. We have also shown that the nadD gene is essential for growth in E. coli.
Stancek
Martin
M
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. martin.stancek@invitro.se
Schnell
Robert
R
Rydén-Aulin
Monica
M
D001665:Binding Sites / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation
D029968:Escherichia coli Proteins; D009612:Nicotinamide-Nucleotide Adenylyltransferase; C038480:nicotinic acid mononucleotide adenylyltransferase