Crystal structure analysis of dipeptidyl amino peptidase from P. mexicana WO24.

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, DAP BII, DAP BIII, and DAP IV (bacterial DPP4), POP from Pseudoxanthomonas mexicana WO24, and demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family and they are classified into the clan SC, family S9 in the MEROPS database. On the basis of the enzymological data we obtained, we proposed that bacterial DAPs should be classified in a manner different from that of mammalian DPPs, except for the DAP IV. The DAP IV liberates dipeptides from the free amino terminus and has a specificity for both proline and hydroxyproline residues in the penultimate position of peptides. Here, we report the first structure of the bacterial DPP IV (P. mexicana WO24 DAP IV) complexed with an inhibitor at 2.2 Å resolution. The subunit structure is composed of two domains, the N-terminal eight-bladed β-propeller domain and the C-terminal alpha/beta/alpha sandwich catalytic domain. These structural features are conserved with clan SC S9 family. However, the N-terminal domain contains a unique helix region that extends over the active site acting as a lid, gating substrate or product access. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique helix region is conserved in some kind of bacterial DPP4s except for mammalian DPP4s and some bacterial DPP4s. Some asaccharolytic and anaerobic bacteria can be used protein or peptides as an energy source. Therefore, these bacteria secrete many types of proteases and peptidases. Especially, the elucidation of degradation mechanisms of collagen, including proline and hydroxyproline, are very important from the point of view of host tissue breakdown in pathogens. Our findings suggest that different ligand recognition mechanisms from the bacterial DPP IV to mammalian DPP4 raise the possibility of an antimicrobial development targeting DPP IV from bacteria.

Crystal Structure analyses of dipeptidyl aminopeptidase BII from P. mexicana WO24.

The peptidase family S46 that contains the dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24 is the only exopeptidase family in clan PA peptidases. Our present phylogenetic and experimental studies indicated that the catalytic triad of DAP BII is composed of His 86, Asp 224 and Ser 657 and implied that unknown large helical domains involved in exopeptidase activity[1]. However, three-dimensional structure of a family S46 peptidase has not yet been reported. Thus, the crystal structure of DAP BII is essential not only to understand the catalytic mechanism of family S46 peptidases but also to clarify the structural origin of the exo-type peptidase activities of these enzymes. Recently, we have successfully crystallized the DAP BII and collected X-ray diffraction data to 2.3 Å resolution from the crystal. This crystal belonging to space group P2₁2₁2₁, with unit-cell parameters a = 76.55 Å, b = 130.86 Å, c = 170.87 Å[2]. Structural analysis by the multi-wavelength anomalous diffraction method is underway[3]. Here, we report the first crystallization and structural analysis of the DAP BII from P. mexicana WO24 as family S46 peptidase. Other enzymes that belong to this family are DPP7 and DPP11 from Porphyromonas gingivalis, DPP11 from Porphyromonas endodontalis (periodontal pathogen) and DPP11 from Shewanella putrefaciens (multidrug resistance associated opportunistic pathogen). These gram-negative bacterial pathogens are known to asaccharolytic. Especially, Porphyromonas gingivalis is known to utilize dipeptides, instead of free amino acids, as energy source and cellular material. Since S46 peptidases are not found in mammals, we expect our study will be useful for the discovery of specific inhibitors to S46 peptidases from these pathogens.

Structural analyses of the two cysteine desulfurases from Aquifex aeolicus

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer to a wide range of acceptor proteins. IscS degrades L-cysteine into L-alanine and a sulfur atom in a pyridoxal 5'-phosphate (PLP) dependent manner. In this reaction, it is essential for a conserved Lys residue of IscS to form Schiff base (the covalent bonding interaction) with PLP. Recent accumulations of genomic information have revealed that some IscS homologues in archaea and thermophilic bacteria lack this invariant Lys. Here we report the crystal structures of two paralogous cysteine desulfurases, the canonical Aa IscS1 and the invariant Lys lacking Aa IscS2, from Aquifex aeolicus. Aa IscS1/Aa IscS2 were overproduced in E. coli, and purified by heat-treatment and several column chromatography, and crystallized. The structure of Aa IscS1 was determined at 2.00 Å (Rcryst= 19.4% and Rfree = 22.0%), and Aa IscS2 at 2.55 Å (Rcryst= 21.8% and Rfree = 27.0%). Overall structures as well as orientations of the residues in the active site were quite similar to each other. In Aa IscS1 the PLP adduct was anchored in the catalytic pocket of Aa IscS1 by the formation of the aldimine Schiff base with the invariant Lys. Whereas in Aa IscS2 the PLP was not seen in the active pocket, since the catalytic Lys was substituted by Leu. Alternatively, an electron density derived from unknown-small molecule was located in the catalytic site of Aa IscS2. The shape of this electron density was completely different from that of PLP. The Bijvoet difference map calculated from data collected at λ=1.7 Å overlapped with the electron density observed in the active site; the unknown-small molecule probably contains such metals as iron atoms. Furthermore, the ICP-MS analysis demonstrated that as-isolated Aa IscS2 harbored the iron atom in the solution state. More recently we obtained the experimental evidences that non-canonical Aa IscS2 was able to form the binary complex with Aa IscU, which is responsible for a scaffold for the assembly of a nascent Fe-S cluster. Base on the structural/biochemcal results, possible physiological functions of two cysteine desulfrurases will be discussed.