Isolation, characterization and molecular three-dimensional structural predictions of metalloprotease from a phytopathogenic fungus, Alternaria solani (Ell. and Mart.) Sor.

• Keratinolytic enzymes (AsK) of MW 24.5 kDa were identified and characterized from Alternaria solani.
• The enzyme activity was strongly inhibited by EDTA, however PMSF and metal ions had little effect.
• Zinc atom present at helix regions (Helix 3, 6, 7: His57, His130, His169 and Cys123).
• Metal-binding region is assembled by His and Leu (middle of a helix 3, 6, 7 regions).
• This is the first report of secreted AsK metalloprotease from A. solani.

The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A. solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0–10.0 and a temperature range of 45–50°C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na+, K+) had little effect. Metal ions such as MgSO4, CaCl2, KCl at 10 mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A. solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5 kDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-β-sheets, and 9-α-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His57, His130, His169, and Cys123). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing.