Expression and purification of aspartate beta-semialdehyde dehydrogenase from infectious microorganisms.
l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in humans, but is necessary for bacterial cell wall biosynthesis, the enzymes in this pathway are potential targets for the development of new antibiotics. The asd gene that encodes for ASA DH has been obtained from several infectious organisms and ligated into a pET expression vector. ASA DHs from Haemophilus influenza, Pseudomonas aeruginosa, and Vibrio cholerae were expressed as soluble proteins in Escherichia coli, while ASA DH from Helicobacter pylori was obtained primarily as inclusion bodies. The V. cholerae genome contains two asd genes. Both enzymes have been expressed and purified, and each displays significant ASA DH activity. The purification of highly active ASA DH from each of these organisms has been achieved for the first time, in greater than 95% purity and high overall yield. Kinetic parameters have been determined for each purified enzyme, and the values have been compared to those of E. coli ASA DH.
Moore
Roger A
RA
Department of Chemistry, University of Toledo, Toledo, Ohio 43606, USA.
Bocik
William E
WE
Viola
Ronald E
RE
D000818:Animals; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006193:Haemophilus influenzae / Q000201:enzymology*; D016480:Helicobacter pylori / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014734:Vibrio cholerae / Q000201:enzymology*
D001223:Aspartate-Semialdehyde Dehydrogenase