Expansion of the active site of the azoreductase from Shewanella oneidensis MR-1 |
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| Affiliation: | 1. Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China;2. Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, 430074, China;3. University of Chinese Academy of Sciences, Beijing, 100049, China;1. Department of Food and Nutrition Sciences, The University of Reading, Reading, United Kingdom;2. BioCentre Facility, The University of Reading, Reading, United Kingdom;3. Department of Food Technology, Universiti Kuala Lumpur - Malaysian Institute of Chemical and Bioengineering Technology, Malaysia;1. Centre for Environmental Sustainability and Water Security (iPASA), Research Institute for Sustainable Environment, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia;2. Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia;3. Department of Environmental Engineering, Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia;1. Information Materials and Intelligent Sensing Laboratory of Anhui Province, Institutes of Physical Science and Information Technology, Anhui University, Hefei, 230601, China;2. School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China;3. Department of Civil and Environmental Engineering, School of Civil and Environmental Engineering, Ningbo University, Ningbo, 315211, China |
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| Abstract: | Azoreductase from Shewanella oneidensis MR-1 (soAzoR) possesses great potential in cleaving azo bond of azo dyes during degradation progress. However, detailed information on interaction of soAzoR with either prosthetic group or substrate remains unavailable, mainly due to the absence of crystallization of soAzoR. In order to unravel these mechanisms, we firstly built the tertiary structure of soAzoR and then computationally predicted the binding mode of FMN, NADH and a model dye, methyl red (MR). Ten residues of soAzoR, which are predicted to participate in ligands binding, were separately substituted for either alanine or phenylalanine to confirm their function. The homologous modeling result reveals soAzoR employs a typical Rossmann fold. In terms of ligand binding modes, the isoalloxazine ring of FMN is stabilized in planar conformation by amino acids in the loop L6 and L9 region. NADH and MR is superposed against the isoalloxazine ring with an angle and the distance from C4 atom of NADH and azo bond of MR to N5 atom of FMN is 4.3 Å and 4.6 Å, respectively. The result of predicted interaction and enzyme kinetic analysis suggests that Asn96, Gly140 and Gly141 are crucial for FMN and MR binding; Tyr119 and Phe161 are more meaningful for NADH binding; Ser16 plays an important role in appropriately binding of both FMN and NADH. |
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| Keywords: | Azoreductase Active site Catalytic mechanism |
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