Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.
Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.
Kanatani
A
A
School of Pharmaceutical Sciences, Nagasaki University.
Masuda
T
T
Shimoda
T
T
Misoka
F
F
Lin
X S
XS
Yoshimoto
T
T
Tsuru
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D000085822:Prolyl Oligopeptidases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D004269:DNA, Bacterial; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; C090703:oligopeptidase B