Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme.
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.
Kabashima
T
T
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki. t-kabashima @cc.nagasaki-u.ac.jp
Kitazono
A
A
Kitano
A
A
Ito
K
K
Yoshimoto
T
T
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
D004791:Enzyme Inhibitors; D012967:Sodium Dodecyl Sulfate; D000626:Aminopeptidases; C022450:prolyl aminopeptidase