| Autor: |
Bora B; a Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India., Biswas AD; b Molecular Modelling and Simulation Lab, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India., Gurung AB; c Department of Biotechnology and Bioinformatics , North Eastern Hill University , Shillong 793022 , Meghalaya , India., Bhattacharjee A; c Department of Biotechnology and Bioinformatics , North Eastern Hill University , Shillong 793022 , Meghalaya , India., Mattaparthi VS; b Molecular Modelling and Simulation Lab, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India., Mukherjee AK; a Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India. |
| Abstrakt: |
Microbial fibrinogenolytic serine proteases find therapeutic applications in the treatment of thrombosis- and hyperfibrinogenemia-associated disorders. However, analysis of structure-function properties of an enzyme is utmost important before its commercial application. In this study, an attempt has been made to understand the structure of a fibrinogenolytic protease enzyme, "Bacifrinase" from Bacillus cereus strain AB01. From the molecular dynamics trajectory analysis, the modelled three-dimensional structure of the protease was found to be stable and the presence of a catalytic triad made up of Asp102, His83 and Ser195 suggests that it is a serine protease. To understand the mechanism of enzyme-substrate and enzyme-inhibitor interactions, the equilibrated protein was docked with human fibrinogen (the physiological substrate of this enzyme), human thrombin and with ten selective protease inhibitors. The Bacifrinase-chymostatin interaction was the strongest among the selected protease inhibitors. The serine protease inhibitor phenyl methane sulphonyl fluoride was found to interact with the Ser134 residue of Bacifrinase. Furthermore, protein-protein docking study revealed the fibrinogenolytic property of Bacifrinase and its interaction with Aα-, Bβ- and Cγ-chains human fibrinogen to a different extent. However, biochemical analysis showed that Bacifrinase did not hydrolyse the γ-chain of fibrinogen. The in silico and spectrofluorometric studies also showed interaction of Bacifrinase with thrombin as well as fibrinogen with a Kd value of 16.5 and .81 nM, respectively. Our findings have shed light on the salient structural features of Bacifrinase and confirm that it is a fibrinogenolytic serine protease. |
