Abstract
Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with kcat/Km of 10.62 ± 2.51 μM−1 s−1, while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing kcat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria.
Highlights
Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobe, is a major causative organism of chronic periodontitis [1,2], which leads to loss of permanent teeth [3,4,5] and, affects quality of life, especially in elderly individuals [6]
Classification of DPPs from DPPI to DPP11 has generally been performed based on differences in substrate specificities [16], in which the P1-position residues seem to be most critical
The previous study on substrate specificity of P. gingivalis DPP7 had focused on the P1 position, but was unable to refer to P2 position due to limited number of substrates used
Summary
Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobe, is a major causative organism of chronic periodontitis [1,2], which leads to loss of permanent teeth [3,4,5] and, affects quality of life, especially in elderly individuals [6]. Much attention has recently been paid to this bacterium because of its close. P. gingivalis does not ferment glucose, cellobiose, lactose or sucrose [10], while it requires proteinaceous substrates as carbon and energy sources. Amino acids are incorporated into the bacterial cells mainly as dipeptides, not as free amino acids [11,12]. Dipeptidylpeptidases (DPPs) that release dipeptides by cleavage of the penultimate peptide bond (P1 position) from the N-terminus of an oligopeptide are crucial for bacterial metabolism [13,14,15]. If dipeptides with any sequences can be released by DPPs, it appears to be advantageous for energy production by the bacterium
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