Prolyl tripeptidyl peptidase (PTP, EC 3.4.14.12) from P. gingivalis is well known to cause periodontitis by degrading tissue collagens. This enzyme was the first studied by Potempa and colleagues (1), and then we solved the crystal structure of PTP (2). Our study revealed that the PTP structure consists of the catalytic and β-propeller domains and a large cavity between these two domains like dipeptidyl peptidase 4 (DPPIV). We also developed a new inhibitor towards PTP (H-Ala-Ile-pyrrolidin-2-yl boronic acid) and clarified the mechanism how PTP recognizes its substrate, using the compound (3).
In this study, we sought for PTP inhibitor in rice bran (rPTPI). rPTPI was purified from soluble protein fractions of rice bran by ammonium sulfate fractionation, followed by hydrophobic, ion exchange and size exclusion chromatographies. A molecular mass of rPTPI was estimated to be 24 kDa by SDS-PAGE under the reducing conditions. The purified rPTPI blocked the peptidase activity of PTP, but not those of DPPIV, prolyl oligopeptidase and prolyl aminopeptidase. As the amino acid sequences of tryptic peptides from rPTPI were determined by mass spectrometric analysis, we found that rPTPI is a Mn-type enzyme of the superoxide dismutase superfamily (rMnSOD) (GeneBank ID: Os05g0323900).
Moreover, a cDNA encoding rice MnSOD was chemically synthesized according to the E. coli codon usage, cloned into pET-32a vector, and then expressed in the E. coli strain BL21(DE3). The resulting recombinant protein, in which rMnSOD was N-terminally tagged with thioredoxin (Trx), exhibited both of the SOD and PTPI activities, whereas Trx alone had neither of them. We confirmed that SOD inhibit PTP by using SODs from different sources ..
.
1. JBC 274, 9246-9252 (1999); 2. JMB 362, 228-240 (2006); 3. JMB 375, 708-719 (2008)