Research article

Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro

Martin Stancek² , Robert Schnell³ and Monica Rydén-Aulin¹

¹  Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden

²  In vitro Sweden AB, Box 21160, S-100 31 Stockholm, Sweden

³  Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

author email corresponding author email

BMC Biochemistry 2005, 6:16doi:10.1186/1471-2091-6-16

Published:	9 September 2005

Abstract

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.

Conclusion

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.