Discrimination based on Gly and Arg/Ser at position 673 between dipeptidyl-peptidase (DPP) 7 and DPP11, widely distributed DPPs in pathogenic and environmental gram-negative bacteria

• Published in: Biochimie 2013-04, Vol.95 (4), p.824-832
• Main Authors: Rouf, Shakh M.A., Ohara-Nemoto, Yuko, Hoshino, Tomonori, Fujiwara, Taku, Ono, Toshio, Nemoto, Takayuki K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arginine; Bacteroides fragilis; Bacteroides vulgatus; Bacteroidetes; Capnocytophaga gingivalis; Dipeptidyl peptidase; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - chemistry; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - metabolism; Environment; Flavobacterium psychrophilum; Gene Expression Regulation, Bacterial; Glycine; Gram-Negative Bacteria - classification; Gram-Negative Bacteria - enzymology; Gram-Negative Bacteria - genetics; Molecular Sequence Data; Phylogeny; Porphyromonas gingivalis; Proteobacteria; S46 peptidase; Serine; Shewanella putrefaciens; Substrate Specificity
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2012.11.019

Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al. (2011) J. Biol. Chem. 286, 38115–38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens. Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity, and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser673 exhibited Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with Arg673 from Bacteroidetes species. Mutagenesis experiments revealed that Arg673/Ser673 were indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was mediated by Gly673. Taken together with the distribution of the two genes, all 264 members of the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A more compelling phylogenic tree based on the conserved C-terminal region suggested two gene duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the genus Bacteroides. ► Expression and site-directed mutagenesis were employed to define S46 members' substrate specificity. ► Gly673 in DPP7 and Arg/Ser673 in DPP11 were critical for the substrate specificity. ► All S46 members were assigned based on the single residue at position 673. ► A new phylogenic tree suggested two gene duplication events in the phylum Bacteroidetes.