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

Abstract

Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapourdiffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2 1 2 1 2 1 , with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å . Structural analysis by the multiwavelength anomalous diffraction method is in progress.

S46 Peptidases are the First Exopeptidases to be Members of Clan PA

“…To obtain peptide-free DAP BII crystals 39 , the samples were crystallised using the hanging-drop method, in which 1 μl of protein solution (10 mg/ml DAP BII in 80 mM Tris-HCl, pH 8.5) was mixed with the same volume of reservoir solution (18% (w/v) PEG8000, 20% (v/v) glycerol, and 2 mM zinc chloride in 0.08 M CHES-NaOH, pH 9.5) and incubated at 293 K. The drops were suspended over 500 μl of reservoir solution in 24-well plates. Peptide-free crystals were also obtained using a counter-diffusion crystallisation method 40 under a microgravity environment in the Japanese Experimental Module “Kibo” at the International Space Station (ISS) 41 .…”

Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

“…Recently, the first three-dimensional structure of a S46 peptidase has been determined, for DAP BII 31 32 . That study revealed that DAP BII is a homodimer and that each subunit contains a peptidase domain including a double β-barrel fold that is characteristic of the chymotrypsin superfamily 33 34 , as well as an unusual α-helical domain that regulates the exopeptidase activity of DAP BII.…”

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl peptidase 11 fromPorphyromonas gingivalis

“…Assuming the presence of one subunit per asymmetric unit led to an empirically acceptable V M value of 2.79 Å 3 Da À1 , corresponding to a solvent content of 55.9% (Matthews, 1968). Since another S46 peptidase, bacterial DAP BII (Ogasawara et al, 1996;Sakamoto, Suzuki, Iizuka, Tateoka, Roppongi, Fujimoto et al, 2014;Sakamoto, Suzuki, Iizuka, Tateoka, Roppongi, Okada et al, 2014), was observed to be a dimer, we assume that PgDPP11 also forms a dimer. In that case, the molecular twofold axis of the dimeric PgDPP11 molecule must coincide with a crystallographic twofold axis of the C222 1 crystal of PgDPP11.…”

“…Initially, we tried to solve the structure of PgDPP11 by molecular-replacement techniques. Structures of DAP BII complexed with or without a peptide (Sakamoto, Suzuki, Iizuka, Tateoka, Roppongi, Okada et al, 2014), belonging to the S46 peptidase family (Suzuki et al, 2014) and having 29% sequence identity with PgDPP11, were used as search models. Since these attempts failed, a search for heavyatom derivatives intended for phasing by the multiple/single isomorphous replacement method and the preparation of selenomethionine-substituted PgDPP11 using LeMaster medium (Hendrickson et al, 1990) and E. coli B834(DE3) cells for phasing by Se-MAD/SAD methods are under way.…”

“…Note that the mammalian DPP7s belonging to the S28 family are not related to the bacterial DPP7s belonging to the S46 family described above. Recently, we have determined the first crystal structure of a clan PA family S46 peptidase, dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24 (Sakamoto, Suzuki, Iizuka, Tateoka, Roppongi, Fujimoto et al, 2014;Sakamoto, Suzuki, Iizuka, Tateoka, Roppongi, Okada et al, 2014). Our study revealed that DAP BII contains a peptidase domain including a typical double -barrel fold, which is characteristic of clan PA peptidases such as chymotrypsin, and a previously unreported large -helical domain.…”