Isolation, biochemical characterization, and primary structure and active site determination of Dioscorea opposita...

A protease was purified to homogeneity from Dioscorea opposita "Nagaimo" using ion exchange, hydrophobic and gel filtration columns, and its biochemical characterization including molecular weight, substrate specificity and kinetic parameters were determined. Protease activity was strongly inhibited by AEBSF, DCI and TLCK. The enzyme moderately inhibited by NEM and HgCl₂. The enzyme activity inhibited by NEM and HgCl₂ was restored with the addition of β-ME. These findings suggest that the enzyme is a trypsin-like serine protease, which is regulated by SH compounds. The N-terminal amino acid of this protease is blocked in an unknown manner. We determined the structure of the cDNA and deduced amino acid sequence of the protease from D. opposita. The cDNA was composed of 2420 nucleotides and encoded 751 amino acids in the coding region. The results indicated that this enzyme is an oligopeptidase B (OPB), consisting of a N-terminal region (M¹ ∼ T4⁷), a N-terminal β-propeller domain (A⁴⁸∼ L⁴⁶⁵), a connecting domain (K⁴⁶⁶ ∼ D⁵²⁷), a peptidase_S9 domain (P⁵²⁸ ∼ D⁷⁴⁴) and C-terminal region (R⁷⁴⁵ ∼ S⁷⁵¹). The overall homology of amino acid sequences of D. opposita to D. alata and D. rotundata was 99.07 % and 97.07 %, respectively. The catalytically active amino acid sites [S⁵⁹⁹, D⁶⁸⁴, and H⁷¹⁹] among these yam species were found to be highly conserved. Site-directed mutagenesis confirmed that these three the active center.