Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24

Abstract

Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyzeGly-Phe-pnitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1 positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.

Purification and Characterization of a Dipeptidyl Carboxypeptidase from Pseudomonas sp. WO24

“…Recently, we have reported the identification, purification, and characterization of novel dipeptidyl aminopeptidases (DAPs) named DAP BI,25) DAP BTI, and DAP BITI, 26) and of a ubiquitous DAP IV 27 ) from Pseudomonas sp. W024.…”

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).…”

“…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).…”

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

“…DPP IV (EC 3.4.14.5), a serine protease with an optimum pH of 8-9, specifically cleaves gly-pro from the N-termini of its substrates. Several DPPs, including some with specificities different from those in mammals, have recently been identified in microorganisms (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) including Dictyostelium discoideum (13)(14)(15)(16).…”

“…DPP II (EC 3.4.14.2), a serine protease with an acidic optimum pH of 4.5-6, preferentially cleaves dipeptides from the N-termini of X-ala-or X-pro-NA. DPP III (EC 3.4.14.4) is a serine protease with an optimum pH of [8][9][10].…”