title: Cupincin: A Unique Protease Purified from Rice (Oryza sativa L.) Bran Is a New Member of the Cupin Superfamily.
abstract: Cupin superfamily is one of the most diverse super families. This study reports the purification and characterization of a novel cupin domain containing protease from rice bran for the first time. Hypothetical protein OsI_13867 was identified and named as cupincin. Cupincin was purified to 4.4 folds with a recovery of 4.9%. Cupincin had an optimum pH and temperature of pH 4.0 and 60 °C respectively. Cupincin was found to be a homotrimer, consisting of three distinct subunits with apparent molecular masses of 33.45 kDa, 22.35 kDa and 16.67 kDa as determined by MALDI-TOF, whereas it eluted as a single unit with an apparent molecular mass of 135.33 ± 3.52 kDa in analytical gel filtration and migrated as a single band in native page, suggesting its homogeneity. Sequence identity of cupincin was deduced by determining the amino-terminal sequence of the polypeptide chains and by and de novo sequencing. For understanding the hydrolysing mechanism of cupincin, its three-dimensional model was developed. Structural analysis indicated that cupincin contains His313, His326 and Glu318 with zinc ion as the putative active site residues, inhibition of enzyme activity by 1,10-phenanthroline and atomic absorption spectroscopy confirmed the presence of zinc ion. The cleavage specificity of cupincin towards oxidized B-chain of insulin was highly specific; cleaving at the Leu15-Tyr16 position, the specificity was also determined using neurotensin as a substrate, where it cleaved only at the Glu1-Tyr2 position. Limited proteolysis of the protease suggests a specific function for cupincin. These results demonstrated cupincin as a completely new protease.
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, India.
authors.forename: Roopesh
authors.initials: R
authors.lastname: Sreedhar
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, India.
authors.forename: Purnima
authors.initials: P
authors.lastname: Kaul Tiku
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D012275:Oryza / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010447:Peptide Hydrolases