Isoforms of dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24: role of the signal sequence and overexpression in Escherichia coli

Identification of the Catalytic Triad of Family S46 Exopeptidases, Closely Related to Clan PA Endopeptidases

“…In the sequence coding for the signal peptide (ATG CGT CCG AAC CTG CTC GCC GCC GCC ATC GCG GTC CCG TTG TCC CTG CTC GCC GCC CAG ATC GCC CAG GCG), multiple occurrences of the same codon for Ala ( GCC ) might, however, cause a problem with translation. As DAP BI, DAP BIII (unpublished result) and DAP IV19 from P. mexicana WO24 could be expressed in E. coli we constructed a chimeric gene that coded for the fusion of the mature DAP BII polypeptide with the signal peptide of DAP BIII. We expressed this fusion gene by means of the lac promoter in JM109 cells and determined the substrate specificity of the enzyme that was purified from cell-free extracts by ammonium sulfate precipitation.…”

“…AB889525) was placed under the control of the lac promoter. Regions from both genes were cloned into a vector used for the heterologous expression of DAP IV, pKF18-k-219. First, a 2.1 kb KpnI-BamHI fragment from pDAP BII with a part of the coding region of dapb2 and some upstream sequence, was ligated into pKF18k-2, yielding pK8b2.…”

“…DAPs are present in all organisms, from bacteria to mammals, and the enzyme contributes to several important physiological functions such as the absorption of nutrients, immunity and the ability of pathogens to invade host cells151617. DAP BI18 and DAP IV19 show amino acid sequence homology and similar substrate specificity to enzymes that belong to the peptidase family S920 in clan SC, such as oligopeptidase B and DPP IV. In contrast, no homologs have been identified for DAP BII and DAP BIII.…”

Periplasmic form of dipeptidyl aminopeptidase IV fromPseudoxanthomonas mexicanaWO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis

“…Dipeptidyl aminopeptidase (DAP or DPP; EC 3.4.14) catalyses the removal of dipeptides from the amino-termini of peptides and proteins. In microorganisms, we have reported the identification, purification and characterization of DAP BI (bacterial dipeptidyl aminopeptidase; Ogasawara, Ochiai et al, 1996;Ogasawara et al, 1998); DAP BII (Ogasawara, Kobayashi et al, 1996;Suzuki et al, 2014;Sakamoto et al, 2014); DAP BIII (Ogasawara, Kobayashi et al, 1996); DAP IV (in this paper, we designate bacterial DPP IV as DAP IV; Ogasawara, Ogawa et al, 1996;Ogasawara et al, 2005) and prolyloligopeptidase (POP; Ogasawara et al, unpublished work) from Pseudoxanthomonas mexicana WO24, which is a Gram-negative aerobic bacteria isolated from the wastewater of a bean-curd (tofu) factory. We have demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family (Kanatani et al, 1991;Rawlings et al, 1991) and that they are classified into clan SC, family S9 in the MEROPS database (Rawlings et al, 2014), whereas DAP BII is classified into the clan PA, family S46 (Suzuki et al, 2014;Sakamoto et al, 2014).…”

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

“…We have reported the identification, purification and characterization of DAP BI (bacterial dipeptidyl aminopeptidase; Ogasawara, Ochiai et al, 1996;Ogasawara et al, 1998), DAP BII (Ogasawara, Kobayashi et al, 1996), DAP BIII (Ogasawara, Kobayashi et al, 1996) and DAP IV (Ogasawara, Ogawa et al, 1996;Ogasawara et al, 2005) from Pseudoxanthomonas mexicana WO24, which is a Gram-negative aerobic bacterium isolated from the wastewater of a bean curd (tofu) factory. 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).…”