Structure of hydrogen sulfide-producing enzyme from a periodontal pathogen

Hydrogen sulfide (H₂S) is one of the predominant volatile sulfur compounds that are primarily responsible for oral malodor and contribute to the progress of periodontal disease. H₂S in the human oral cavity is generally produced by enzymatic actions of oral bacteria. Fusobacterium nucleatum, a Gram negative periodontal pathogen, is known to be one of the heaviest H₂S producers [1]. For now, four genes (fn0625, fn1055, fn1220, and fn1419) encoding pyridoxal-5′-phosphate (PLP)-dependent H₂S-producing enzymes have been identified and characterized in F. nucleatum ATCC 25586. Of the four enzymes, Fn1055 protein is a unique H₂S-producing enzyme, which produces H₂S and L-serine from L-cysteine [2]. Therefore, Fn1055 might play important roles in L-serine biosynthesis in addition to H₂S production in this periodontal pathogen. Crystal structures of recombinant Fn1055 and its site-directed mutant complex with L-cysteine (a substrate) were determined at 2.1 Å resolution. The enzyme forms a homodimer whose subunits are related by a two-fold axis. The subunit is composed of two domains with α/β structure. The PLP cofactor forms a covalent internal aldimine linkage with the ε-amino group of Lys46 at the bottom of active site cleft between the domains, in the absence of substrate. On the other hand, in the cocrystal of mutant with L-cysteine, the introduced L-cysteine was found to be covalently bound to PLP, instead of Lys46. This covalent intermediate was identified as an α-aminoacrylate, which is the key species of PLP-dependent-enzyme catalysis, by spectrophotometric measurement. Along with the intermediate formation, closure of active site cleft was also observed. Furthermore, we found an amino acid residue acting as a base and confirmed its indispensability for catalysis by enzymatic analyses. These results support that H₂S production by Fn1055 proceeds through the β-elimination of L-cysteine, and enable us to propose a detailed catalytic mechanism of Fn1055.

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.