Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme.
The pyroglutamyl peptidase [EC 3.4.11.8] gene from Bacillus amyloliquefaciens was cloned and expressed in Escherichia coli DH1. The transformant of E. coli DH1 harboring plasmid pBPG 1 with a 2.1 kb chromosomal DNA fragment showed 80-fold higher activity than B. amyloliquefaciens. The nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chain-termination method. An open reading frame of 648 bp starting with an ATG methionine codon was found, which encodes a protein of 215 amino acid residues with a deduced molecular weight of 23,286. The enzyme has two cysteine residues (Cys68 and Cys144) per subunit molecule. Substitution of Cys144 with Ser by site-directed mutagenesis resulted in a complete loss of the activity, while that of Cys68 with Ser did not affect the activity at all. This result and titration with DTNB suggest that Cys144 is concerned in the catalytic action and Cys68 is located inside the enzyme. The expressed enzyme was purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with an activity recovery of 42.7%. The enzyme was most active at pH 6.5 and stable at pH 7.0-9.0. Its molecular weight was estimated to be 51,000 by gel filtration, suggesting it to be a dimer. Big crystals of the wild and PCMB-modified enzymes were obtained by the hanging drop method.
Yoshimoto
T
T
School of Pharmaceutical Sciences, Nagasaki University.
Shimoda
T
T
Kitazono
A
A
Kabashima
T
T
Ito
K
K
Tsuru
D
D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping
D000596:Amino Acids; D004269:DNA, Bacterial; D011751:Pyroglutamyl-Peptidase I; D003545:Cysteine