Isolation, biochemical characterization, and primary structure and active site determination of Dioscorea opposita (‘Nagaimo’) oligopeptidase B

TL;DR

This paper reports the first isolation and characterization of a trypsin-like serine protease, oligopeptidase B, from the plant Dioscorea opposita, including its structure and active site.

Contribution

The first isolation and biochemical characterization of oligopeptidase B from Dioscorea opposita, along with its cDNA structure and active site identification.

Findings

The enzyme is a trypsin-like serine protease regulated by SH compounds.

The protease is identified as oligopeptidase B with a conserved active site across yam species.

Site-directed mutagenesis confirmed the catalytic triad S599, D684, and H719.

Abstract

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.

Image 1

•A Protease was purified from D. opposita (Nagaimo) for the first time.•The biochemical properties of the purified protease were clarified, and it was found to be a trypsin-type serine protease.•Determination of the internal amino acid sequence of the protease revealed that this protease is Oligopeptidase B.•The cDNA structure of this protease was first elucidated.•The active center of this enzyme was identified using site-directed mutagenesis.

A Protease was purified from D. opposita (Nagaimo) for the first time.

The biochemical properties of the purified protease were clarified, and it was found to be a trypsin-type serine protease.

Determination of the internal amino acid sequence of the protease revealed that this protease is Oligopeptidase B.

The cDNA structure of this protease was first elucidated.

The active center of this enzyme was identified using site-directed mutagenesis.

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