title: Acyl-CoA reductase PGN_0723 utilizes succinyl-CoA to generate succinate semialdehyde in a butyrate-producing pathway of Porphyromonas gingivalis.
abstract: The molecular basis of butyrate production in Porphyromonas gingivalis has not been fully elucidated, even though butyrate, a short chain fatty acid (SCFA), can exert both beneficial and harmful effects on a mammalian host. A database search showed that the amino acid sequence of PGN_0723 protein was 50.6% identical with CoA-dependent succinate semialdehyde dehydrogenase (SSADH) in Clostridium kluyveri. By contrast, the protein has limited identity (19.1%) with CoA-independent SSADH in Escherichia coli. Compared with the wild type, growth speed, and final turbidity were lower in the PGN_0723 deletion strain that was constructed by replacing the PGN_0723 gene with an erythromycin resistance cassette. Gas chromatography mass spectrometry revealed the supernatant concentrations of the SCFAs butyrate, isobutyrate, and isovalerate, but not propionate, in the PGN_0723 deletion strain were also lower than those in the wild type. The wild-type phenotype was restored in a complemented strain. We cloned the PGN_0723 gene, purified the recombinant protein, and computationally constructed its three-dimensional model. A colorimetric assay and liquid chromatography-tandem mass spectrometry analysis demonstrated that the recombinant PGN_0723 produces succinate semialdehyde, which is an intermediate in the P. gingivalis butyrate synthesis pathway, not from succinate but from succinyl-CoA in the presence of NAD(P)H via a ping-pong bi-bi mechanism. Asn110Ala and Cys239Ala mutations resulted in a significant loss of the CoA-dependent PGN_0723 enzymatic activity. The study provides new insights into butyrate production, which constitutes a virulence factor in P. gingivalis.
authors.affiliation: Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan. Electronic address: yasuoy@dpc.agu.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan; Department of Removable Prosthodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
authors.forename: Mitsunari
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Structural Biology, School of Pharmacy, Iwate Medical University, Yahaba, Iwate, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Kezuka
authors.affiliation: Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Hasegawa
authors.affiliation: Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nagano
authors.affiliation: Department of Removable Prosthodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Takebe
authors.affiliation: Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
authors.forename: Fuminobu
authors.initials: F
authors.lastname: Yoshimura
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002087:Butyrates / Q000378:metabolism*; D017353:Gene Deletion; D020125:Mutation, Missense; D009249:NADP / Q000235:genetics / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics; D050604:Succinate-Semialdehyde Dehydrogenase (NADP+) / Q000235:genetics / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D002087:Butyrates; D009249:NADP; C012046:succinyl-coenzyme A; D050604:Succinate-Semialdehyde Dehydrogenase (NADP+)
keywords: Butyrate; Porphyromonas gingivalis; Short chain fatty acid; Succinate semialdehyde; Succinyl-CoA