title: Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme.
abstract: We cloned and sequenced the Serratia marcescens prolyl aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36,083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36,000 and 38,000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3,4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a= b =65.6 A, and c=169.8 A, a space group P4(1)2(1)2 or P4(3)2(1)2, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 A resolution.
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University, Nagasaki. t-kabashima @cc.nagasaki-u.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Kabashima
authors.forename: A
authors.initials: A
authors.lastname: Kitazono
authors.forename: A
authors.initials: A
authors.lastname: Kitano
authors.forename: K
authors.initials: K
authors.lastname: Ito
authors.forename: T
authors.initials: T
authors.lastname: Yoshimoto
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate
chemical_list: D004791:Enzyme Inhibitors; D012967:Sodium Dodecyl Sulfate; D000626:Aminopeptidases; C022450:prolyl aminopeptidase