title: Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium.
abstract: Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E. coli and the K(m) values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E. coli and S. typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Farida
authors.initials: F
authors.lastname: Khan
authors.forename: Venkatakrishna R
authors.initials: VR
authors.lastname: Jala
authors.forename: N Appaji
authors.initials: NA
authors.lastname: Rao
authors.forename: H S
authors.initials: HS
authors.lastname: Savithri
mesh_terms: D000642:Ammonia-Lyases / Q000737:chemistry*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000737:chemistry*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D000642:Ammonia-Lyases; C023147:diaminopropionate ammonia-lyase; D003545:Cysteine