A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24

Abstract

An activity similar to that of dipeptidyl aminopeptidase I (DAP I) which releases dipeptide from Gly-Argp-nitroanilide (Gly-Arg-pNA) was detected in a Pseudomonas sp. An enzyme was isolated and purified about 400-fold by a series of column chromatographies. The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer. The enzyme had an isoelectric point of 4.7. It is optimally active at pH 9.0. The K m and V max of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 mol/min/mg, respectively. The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-␤-naphthylamide (Arg-Arg-MNA), a model substrate for DAP III. The K m and V max for Arg-Arg-MNA were 0.019 mM and 145 mol/min/mg, respectively. This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiotensins I and II. The enzyme activity was completely inhibited by Zn(II) (0.5 mM), tosyl-L-Lys-chloromethyl ketone (0.1 mM), and leupeptin (0.1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease inhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphate) and thiol protease inhibitors. The substrate specificity, classification of catalytic site, and other enzymatic properties demonstrate that this enzyme is distinct from the previously described mammalian DAPs I and III and Saccharomyces cerevisiae DAP III. These results indicate that DAP BI may be a new type of the DAP family.Dipeptidyl aminopeptidase (DAP) is capable of sequentially removing dipeptides from the amino termini of peptides and proteins. In mammalian cells, four distinct enzymes with DAP activity have been purified from the liver, brain, and pituitary on the basis of substrate specificity by using unique naphthylamide (NA) substrates, specific pH optima, and catalytic classification (13, 15). DAP I (cathepsin C) cleaves Gly-Arg-␤-naphthylamide (Gly-Arg-␤NA) and Gly-Phe-␤NA at pH 6, and DAP II cleaves Lys-Ala-␤NA with a pH optimum of 5. DAP III degrades Arg-Arg-␤NA with a pH optimum of 8 to 9, while DAP IV hydrolyzes Gly-Pro-␤NA at pH 8. DAP I is a lysosomal cysteine protease belonging to the papain superfamily. It possesses a broad specificity (20) for liberating dipeptides whose amino-terminal amino acid is not basic (Arg or Lys) and whose penultimate amino acid is not proline and that are not linked to proline through a peptide bond. DAP I appears to play an important role not only in intracellular protein degradation, but also in cell growth and processing and activation of several enzymes (18). On the other hand, DAP III shows relatively narrow substrate specificity on dipeptide NAs but a wide range of activity on peptide substrates. In particular, DAP III pre...

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

“…We previously reported the identification, purification, and characterization of a novel DAP that we named DAP BI (DAP from bacteria, type I) from Pseudomonas sp. strain WO24 (20). Purified DAP BI hydrolyzed GlyArg-pNA but not Gly-Phe-pNA, both of which are model substrates for DAP I, and possesses a strong activity to hydrolyze Arg-Arg-MNA, a general substrate for DAP III.…”

“…We chose Gly-Phe-pNA rather than Gly-Phe-␤NA as the substrate in the routine assay because ␤-naphthylamine is carcinogenic and not suitable for routine use for assaying the enzyme (10). DAP BII and DAP BIII activities were determined from the amounts of released pNA from Gly-PhepNA according to a method reported previously (20). The incubation mixture consisted of 100 l of 3 mM substrate, 500 l of 0.1 M Tris-HCl buffer (pH 8.0 for DAP BII and pH 9.0 for DAP BIII), 300 l of water, and 100 l of an appropriately diluted enzyme solution, and the reaction was initiated by the addition of the enzyme solution.…”

“…Enzymatic activity was also analyzed with derivatives of ␤NA, 4-methoxy-␤NA, and MCAs as substrates (24) according to previously reported methods (20), except for the assay pH (pH 8.0 for DAP BII and pH 9.0 for DAP BIII). One unit of DAP activity was defined as the amount of enzyme that liberates 1 mol of ␤NA, MNA, or MCA per min at 37ЊC.…”

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).…”

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).…”