Crystal structures of a bacterial dipeptidyl peptidase IV reveal a novel substrate recognition mechanism distinct from that of mammalian orthologues.

Dipeptidyl peptidase IV (DPP IV, DPP4, or DAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position. The substrate recognition mechanism has been fully elucidated for mammalian DPP IV by crystal structure analyses but not for bacterial orthologues. Here, we report the crystal structures of a bacterial DPP IV (PmDAP IV) in its free form and in complexes with two kinds of dipeptides as well as with a non-peptidyl inhibitor at 1.90 to 2.47 Å resolution. Acyl-enzyme intermediates were observed for the dipeptide complexes of PmDAP IV, whereas tetrahedral intermediates were reported for the oligopeptide complexes of mammalian DPP IVs. This variation reflects the different structural environments of the active site Arg residues, which are involved in the recognition of a substrate carbonyl group, of mammalian and bacterial enzymes. A phylogenetic analysis revealed that PmDAP IV is a closer relative of dipeptidyl peptidases 8 and 9 (DPP8 and DPP9, DPP IV-family enzymes) than DPP IV. These results provide new insights into the substrate recognition mechanism of bacterial DAP IVs and may assist in the development of selective inhibitors for DAP IVs from pathogenic asaccharolytic bacteria, which utilise proteins or peptides as an energy source.

Periplasmic form of dipeptidyl aminopeptidase IV from Pseudoxanthomonas mexicana WO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis.

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2'…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Šresolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84 Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

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

The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double ß-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms.

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl peptidase 11 from Porphyromonas gingivalis.

Dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) preferentially cleaves substrate peptides with Asp and Glu at the P1 position [NH2-P2-P1(Asp/Glu)-P1'-P2'...]. For crystallographic studies, PgDPP11 was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.82 Å resolution were collected from an orthorhombic crystal form belonging to space group C2221, with unit-cell parameters a = 99.33, b = 103.60, c = 177.33 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

S46 peptidases are the first exopeptidases to be members of clan PA.

The dipeptidyl aminopeptidase BII (DAP BII) belongs to a serine peptidase family, S46. The amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those of clan PA endopeptidases, such as chymotrypsin. However, the molecular mechanism of the exopeptidase activity of family S46 peptidase is unknown. Here, we report crystal structures of DAP BII. DAP BII contains a peptidase domain including a typical double ß-barrel fold and previously unreported α-helical domain. The structures of peptide complexes revealed that the α-helical domain covers the active-site cleft and the side chain of Asn330 in the domain forms hydrogen bonds with the N-terminus of the bound peptide. These observations indicate that the α-helical domain regulates the exopeptidase activity of DAP BII. Because S46 peptidases are not found in mammals, we expect that our study will be useful for the design of specific inhibitors of S46 peptidases from pathogens.

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

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 vapour-diffusion 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 multi-wavelength anomalous diffraction method is in progress.