Protease II from Escherichia coli: Sequencing and Expression of the Enzyme Gene and Characterization of the Expressed Enzyme1

Abstract

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.

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24

“…On the basis of the enzymological data that we obtained, we proposed that bacterial DAPs should be classified in a manner different from that of mammalian DPPs, except for DAP IV (Ogasawara, Kobayashi et al, 1996). We have also characterized a prolyl oligopeptidase (POP) from P. mexicana WO24 (unpublished data) and demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family (Kanatani et al, 1991;Rawlings et al, 1991) and are classified into clan SC, family S9 in the MEROPS database (Rawlings et al, 2012). Recently, we have successfully determined the complete nucleotide sequence of DAP BII from P. mexicana WO24 (accession No.…”

Assessment of Substrate Inhibition of Bacterial Oligopeptidase B

“…The exact physiological function(s) and the physiological substrates of the OPB are unknown. Although the OPB was first cloned and characterized from Escherichia coli 4,9) and Moraxella lacunata, 6) bacterial OPBs have received much less attention than their homologues from protozoa. Thus their 3D structures and roles in bacterial virulence have not been clarified.…”

“…Arginine-or lysine-containing peptides of no more than about 30 residues can be hydrolyzed, [4][5][6] since the N-terminal β-propeller domain of the enzymes blocks access of large globular proteins to the catalytic machinery. 3) However, it has been demonstrated that OPB can cleave, in addition to low-molecular-mass peptides, several basic proteins in a restricted fashion, including human histones H1, H2A, H3, and H4.…”

Prolylendopeptidase Inhibitory Activity of a Glial Fibrillary Acidic Protein Fragment and Other Proline-rich Peptides

“…[1][2][3][4][5] This enzyme is widely distributed in various organs, particularly in the brain, skeletal muscle, and testis of mammals. A PEP inhibitory peptide, GFAP-(38-55) (MPPP-LPARVOFSLAGALN, a fragment of glial fibrillary acidic protein, K j = 8.6 pM), was previously isolated from bovinc brain.…”