Evolutionary Trace Analysis of Azoreductase at the Ligand Binding Site and Enhancing the Active Site through Site Directed Mutagenesis

Shobana S

doi:10.4172/jpb.1000240

Abstract

Azoreductases belong to the family of oxidoreductases and are characterized with detoxification of azo dyes. Azoreductases are involved in bioremediation, to eliminate the color effluents found in waste waters. In this study, the effectiveness of azoreductase in bioremediation was analysed, and the aim was to find a way to increase the efficiency of the enzyme towards toxic compound degradation, more rapidly. To estimate this, the structure function relationship was determined by evolutionary trace (ET) analysis at ligand binding site. The class specific site of ET analysis was mapped with the crystal structure of azoreductase (1NNI). Site directed mutagenesis was performed at the residues, in very close proximity to the active site residues, hydrogen bonded to ligand. Based on the work done, it was found that Glycine at 106th position plays a crucial role in enzyme activity.

Highlights

The ease of synthesis and chemical stability of azo dyes possessing one or more azo groups helps them to be widely used in textile, printing, cosmetics, pharmaceutical, food and many other industries [1]
Azoreductase catalyses the reductive cleavage of the azo bond, and acts as the key enzyme expressed in all azo dye degrading bacteria [2]
In this protein (1NNI), four residues namely Asp (33), Tyr (74), His (75), and Lys (83) were identified as catalytic residues, where Tyr (74) and Lys (83) play a crucial role in azobenzene reductase extracted from Bacillus subtilis [7]

Summary

Introduction

The ease of synthesis and chemical stability of azo dyes possessing one or more azo groups helps them to be widely used in textile, printing, cosmetics, pharmaceutical, food and many other industries [1]. Bacillus subtilis is considered to be a widely spread bacterium, commonly recovered from water, soil, air and decomposing plant residue This bacterium produces an endospore, which allows it to overcome extreme conditions of heat and drying up in the environment. Olivier Lichtarge in 1996 developed the evolutionary trace (ET) analysis [8] This method depends on both sequence and structural information in order to analyze functional sites of a protein or group of proteins. It determines the conserved amino acid residues in an alignment, and the information is mapped onto known 3D protein structures. The best-ranked residues are clustered spatially in the protein structure and thereby reveal the location of functional sites [9]

Methodology

Results and Discussion

Discussion