title: Kinetic characterization of arginine deiminase and carbamate kinase from Streptococcus pyogenes M49.
abstract: Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in Escherichia coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (e.g., ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12mM (mean±SD) and 1.51±0.07μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08mM and 1.10±0.10μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively.
authors.affiliation: Rostock University Medical Centre, Institute of Medical Microbiology, Virology, and Hygiene, Schillingallee 70, 18057 Rostock, Germany.
authors.forename: Silvio
authors.initials: S
authors.lastname: Hering
authors.forename: Antje
authors.initials: A
authors.lastname: Sieg
authors.forename: Bernd
authors.initials: B
authors.lastname: Kreikemeyer
authors.forename: Tomas
authors.initials: T
authors.lastname: Fiedler
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D006867:Hydrolases; C019723:arginine deiminase
keywords: Arginine deiminase; Carbamate kinase; LAB; Lactic acid bacteria; Streptococcus pyogenes