title: Purification and characterization of an ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, from Pseudomonas putida 77.
abstract: N-Methylhydantoin amidohydrolase, an ATP-dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70-fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77. The enzyme has a relative molecular mass of 300,000. It is a tetramer of two identical small subunits (M(r) 70,000) and two identical large subunits (M(r) 80,000). The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K+, NH4+, Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The Km and Vmax values for N-methylhydantoin were 32 microM and 9.0 mumol.min-1.mg protein-1. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, DL-5-methylhydantoin, glutarimide and succimide in addition to N-methylhydantoin. 2-Pyrrolidone, 2-oxazolidone, delta-valerolactam, 2,4-thiazolidinedione, 2-imidazolidone, D-5-oxoproline methyl ester, DL-5-oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e. dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self-hydrolysis.
authors.affiliation: Department of Agricultural Chemistry, Kyoto University, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Ogawa
authors.forename: J M
authors.initials: JM
authors.lastname: Kim
authors.forename: W
authors.initials: W
authors.lastname: Nirdnoy
authors.forename: Y
authors.initials: Y
authors.lastname: Amano
authors.forename: H
authors.initials: H
authors.lastname: Yamada
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000581:Amidohydrolases / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D003460:Crystallization; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016958:Pseudomonas putida / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D000255:Adenosine Triphosphate; D000581:Amidohydrolases