Keitarou Saiki

Biosynthesis and functional role of haem O and haem A

Haem O and/or haem A are specifically synthesized for the haem‐copper respiratory oxidases. A 17‐carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen‐reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB‐ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.

Heme O biosynthesis in Escherichia coli: The cyoe gene in the cytochrome BO operon encodes a protoheme IX farnesyltransferase

The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme O present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme O. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel enzyme, protoheme IX farnesyltransferase, essential for heme O biosynthesis.

Identification of a Porphyromonas gingivalis novel protein sov required for the secretion of gingipains.

Gingipains are extracellular proteases important for the virulence of Porphyromonas gingivalis; however, the mechanism for the secretion of gingipains is poorly understood. In this report, we found that insertion mutants for PG0809 (83K1 and 83K2) were defective in black pigmentation and hemolysis. We cloned and sequenced PG0809 and found that PG0809 contains two additional nucleotides that are not deposited in the W83 genome database. The revised sequence reveals an in‐frame fusion of PG0810 and PG0809 and is designated the sov gene. We constructed a sov deletion mutant (83K3) and showed that 83K3 was defective in the activities of black pigmentation, hemolysis, and hemagglutination. Furthermore, in 83K3, the activities of gingipains were severely reduced whereas those of other secreted proteases DPPIV, DPP‐7, and PtpA were not affected. Immunoblot analysis using anti‐RgpB antiserum showed that Arg‐gingipains were poorly secreted in an outer membrane or into an extracellular portion but accumulated within the cells of 83K3, suggesting the secretion of gingipains is defected in 83K3. Taken together, our findings indicated that Sov is a novel protein required for the secretion of gingipains and suggested that the secretion system for gingipains is different from the conserved secretion systems.

PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system.

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.

The role of Sov protein in the secretion of gingipain protease virulence factors of Porphyromonas gingivalis

Porphyromonas gingivalis transports Arg-gingipains and Lys-gingipain across the outer membrane via an unknown pathway. Recently, we found that the sov gene of P. gingivalis W83 was required for this step. In the present study, we characterized the Sov protein. We constructed a P. gingivalis strain that expresses histidine-tagged Sov instead of Sov. Subcellular fractionations and a histidine-tag pulldown experiment showed that histidine-tagged Sov was present in an outer membrane fraction. Furthermore, antiserum raised against the terminal regions of Sov obstructed the secretion of Arg-gingipains from wild-type W83 cells. A deletion study showed that the region from Phe2495 to the C-terminus Gln2499 of Sov is essential for gingipain secretion. Anti-histidine-tag immunoglobulins interfered with the secretion of Arg-gingipains by P. gingivalis cells that expressed histidine-tagged Sov. In conclusion, we found that Sov is an outer membrane protein participating in the secretion of gingipains and that the C-terminal region of Sov is exposed to the extracellular milieu and involved in the modulation of Sov function.

Identification of a novel Porphyromonas gingivalis outer membrane protein, PG534, required for the production of active gingipains

Gingipains are secreted endopeptidases important for the virulence and proliferation of Porphyromonas gingivalis; however, their secretion and biogenesis process is not yet fully elucidated. The PG0534 gene of P. gingivalis W83 encodes a novel protein, PG534, of unknown function. In a PG0534 deletion mutant 83K25, the activities of Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp) were reduced to 4-22% of those of the wild-type W83, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. This indicates that PG534 is required for the gingipain activity. Immunoblot analysis using anti-Rgp or anti-Kgp antiserum showed that abnormal forms of gingipains were detected in the extracellular fraction from 83K25, suggesting that 83K25 exhibits dysfunctional gingipain secretory activity. Normal carbohydrate biogenesis of lipopolysaccharide is required for production of the active gingipains; however, lipopolysaccharide was not deficient in 83K25. Subcellular fractionation and immunoblot analysis using anti-PG534 antiserum localized PG534 to the outer membrane. In conclusion, we identified PG534 as a novel outer membrane protein required for the biogenesis of gingipains.

Porphyromonas gingivalis C‐terminal signal peptidase PG0026 and HagA interact with outer membrane protein PG27/LptO

Outer membrane protein PG27 is essential for secretion/maturation of conserved C-terminal domain (CTD) proteins such as gingipains, HagA, and PG0026. To determine the binding partner(s) of PG27, we used a Porphyromonas gingivalis mutant strain, 83K48, which expressed functional histidine-tagged PG27. Purification of histidine-tagged PG27 from 83K48 found that 136-kDa and 264-kDa proteins accompanied histidine-tagged PG27. Mass spectrometry revealed the 136-kDa protein and 264-kDa protein to be PG0026 and PG1837 (HagA), respectively. PG0026 is a C-terminal signal peptidase which cleaves the CTDs of other CTD proteins. A cross-linking and a native electrophoresis studies suggested the interaction between histidine-tagged PG27 and HagA and the interaction between histidine-tagged PG27 and PG0026. We constructed Porphyromonas gingivalis gene disrupting mutants, and characterized them. PG0026 was required for the full activities of gingipains, whereas HagA was not. A mutation disrupting PG0026 affected localization of PG27, but a mutation disrupting PG1837 showed little effect on the expression and localizations of PG27 and PG0026. To determine the functional role of HagA, we used PG1837-disruptant 83K54 which expressed functional histidine-tagged PG27. Histidine-tagged PG27 in 83K54 was co-purified with not only PG0026 but also several contaminated proteins. These results suggest that PG0026 interacts with PG27 in the absence of HagA, and that the binding state of a PG27-PG0026 complex was affected and stabilized by HagA. Taken together, these data suggest that secreted PG0026 anchors to the cell by interacting with PG27, is stabilized by HagA, and functions in processing of other CTD proteins such as gingipains.

An Escherichia coli cyoE gene homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase.

The cyoE gene of the Escherichia coli bo‐type quinol oxidase operon (cyo ABCDE) has been previously shown to encode heme O synthase. To demonstrate a catalytic role of a cyoE homologue (the caaE gene) in the gene cluster for caa 3‐type cytochrome c oxidase of thermophilic Bacillus PS3, we have carried out genetic complementation analysis using the chimeric operon cyo ABCD‐caaE and heme O synthase assay using the CaaE‐overproduced E. coli membranes. We found that the caaE gene encodes a thermotolerant heme O synthase which provides an intermediate for heme A biosynthesis.

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

Background: Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes. Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 μm and 11.02 μm−1 s−1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.

Analysis of Porphyromonas gingivalis PG27 by deletion and intragenic suppressor mutation analyses.

PG27 is required for secretion of virulence factor gingipains, and has recently been proposed as LptO, which is involved in O-deacylation of lipopolysaccharide. In the present study, a predicted 14 anti-parallel β-strand structure of PG27 was ascertained. Deletion study showed that the region from Asp382 to the C-terminal His391 of PG27 is dispensable for the function of PG27. Analysis of C-terminal deletion mutants revealed that the region in strand S14 (Asn369-Gly385) is important for activity. Of the gingipain-defective mutants, ΔThr378-His391 and ΔPhe377-His391 produced amounts of PG27 comparable to those produced by wild-type cells, suggesting that Thr378-Phe381 contains essential residues for the function of PG27. In contrast, ΔPhe381-His391, ΔAla380-His391, ΔLeu379-His391 and ΔArg376-His391 produced no detectable PG27. The defects of the ΔAla380-His391 mutant were suppressed by changing either Ala346 or Ala359 of PG27 to valine. Importantly, Ala346 and Ala359 are located close to Leu379 in the structural model of PG27. A359V compensated for the instability of PG27, but not the gingipain-defective phenotypes, of other deletion mutants tested, suggesting that Ala380 and Phe381 of PG27 are important for the stability of PG27. Lastly, we found that the C-terminal region of PG27 may be located in the periplasm. Taken together, these findings fit well with a predicted β-barrel structure model for PG27, and show that strand S14 is important for its function.