Computational Structural and Functional Characterization of Azorhizobium caulinodans NifH Protein and Associated Cluster Analysis

Abstract

Nitrogenase iron protein, a key enzyme in biological nitrogen fixation, is required for turning atmospheric nitrogen into a physiologically usable form. In the present work, nitrogenase iron protein (encoded by the nifH gene; WP_012169572.1) of Azorhizobium caulinodans, a nitrogen-fixing bacteria recognized for its symbiotic relationship with leguminous plants, was protein of interest. The nitrogenase iron proteins cluster with NifH were identified computationally and characterized using various tools. An in silico model prediction was also done for functional and structural classification including disulphide bonds analysis, molecular docking and other structural bioinformatics analysis. NifH or frxC family signatures (PS00746: ESGGPEPGVGCAG and PS00692: DVLGDVVCGGFAMP). The formation of disulphide bonds between cysteine residues Cys99 and Cys133were recognized to play an important role in protein stability. Potential Fe-S cluster binding site within the nitrogenase iron amino acid sequences Cys99, Cys133 and Gly135 were identified. In docking analysis the residues Gly14-Thr20, Asp41, Lys43, Asp45, Arg48, Asn186, Arg188, Val212-Asp215, Val218, Gln219, Glu222, Gln237 and Tyr241 were interacted with Adenosine-5’-Diphosphate (DrugBank ID: DB16833/ Pharos: CHEMBL14830/ PubChem: 6022/ ChEMBL: CHEMBL14830/ ChEBI: CHEBI:16761). In this interaction, Gly14, Ile15, Gly16, Lys17, Ser18 and Lys43 residues were identified as active site residues. Furthermore, molecular dynamics simulation was performed to evaluate the protein’s conformational stability and compactness with simulation time 300 ns. Also the impact of disulphide bond formation of NifH model on its overall structural integrity was also evaluated. Total 30 potential disulphide bonds were identified using BIOVIA Discovery Studio 2019. The in silico findings shed light on the stabilizing impact of disulphide bonds in the nitrogenase iron protein.