title: Isolation, characterization and molecular three-dimensional structural predictions of metalloprotease from a phytopathogenic fungus, Alternaria solani (Ell. and Mart.) Sor.
abstract: 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: His(57), His(130), His(169), and Cys(123)). 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.
authors.affiliation: Department of Bioresource and Food Science, Konkuk University, 120 Neundong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea; Department of Plant Science, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620024, Tamil Nadu, India.
authors.forename: Murugesan
authors.initials: M
authors.lastname: Chandrasekaran
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, 238 Burt Hall, Manhattan, KS 66506, USA.
authors.forename: Raman
authors.initials: R
authors.lastname: Chandrasekar
authors.affiliation: Department of Bioresource and Food Science, Konkuk University, 120 Neundong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Se-Chul
authors.initials: SC
authors.lastname: Chun
authors.affiliation: Department of Plant Science, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620024, Tamil Nadu, India. Electronic address: sathiyabamam@yahoo.com.
authors.forename: Muthukrishnan
authors.initials: M
authors.lastname: Sathiyabama
mesh_terms: D000528:Alternaria / Q000201:enzymology*; D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010802:Phylogeny; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D010664:Phenylmethylsulfonyl Fluoride; D004492:Edetic Acid; D045726:Metalloproteases; D015032:Zinc
keywords: Alternaria solani; Homology modeling; Matrix-assisted laser desorption and ionization time of flight/mass spectrometry; Metalloprotease; Protease production; Protease purification