Yoshida Yasuo, Sato Mitsunari, Kezuka Yuichiro, Hasegawa Yoshiaki, Nagano Keiji, Takebe Jun, Yoshimura Fuminobu
Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan; Department of Removable Prosthodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.
Arch Biochem Biophys. 2016 Apr 15;596:138-48. doi: 10.1016/j.abb.2016.03.014. Epub 2016 Mar 21.
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.
牙龈卟啉单胞菌中丁酸盐产生的分子基础尚未完全阐明，尽管短链脂肪酸（SCFA）丁酸盐可对哺乳动物宿主产生有益和有害影响。数据库搜索显示，PGN_0723蛋白的氨基酸序列与克氏梭菌中依赖辅酶A的琥珀酸半醛脱氢酶（SSADH）的序列一致性为50.6%。相比之下，该蛋白与大肠杆菌中不依赖辅酶A的SSADH的序列一致性有限（19.1%）。与野生型相比，通过用红霉素抗性盒替换PGN_0723基因构建的PGN_0723缺失菌株的生长速度和最终浊度较低。气相色谱-质谱分析显示，PGN_0723缺失菌株中丁酸盐、异丁酸盐和异戊酸盐（而非丙酸盐）的SCFA上清液浓度也低于野生型。在互补菌株中恢复了野生型表型。我们克隆了PGN_0723基因，纯化了重组蛋白，并通过计算构建了其三维模型。比色测定和液相色谱-串联质谱分析表明，重组PGN_0723通过乒乓双底物机制在NAD(P)H存在的情况下从琥珀酰辅酶A而非琥珀酸产生琥珀酸半醛，琥珀酸半醛是牙龈卟啉单胞菌丁酸盐合成途径中的中间体。Asn110Ala和Cys239Ala突变导致依赖辅酶A的PGN_0723酶活性显著丧失。该研究为丁酸盐的产生提供了新的见解，丁酸盐是牙龈卟啉单胞菌中的一种毒力因子。
