Functional and structural analyses for MlrC enzyme of Novosphingobium sp. THN1 in microcystin-biodegradation: Involving optimized heterologous expression, bioinformatics and site-directed mutagenesis

Enzymatic function of MIrC from Novosphingobium sp. THN1 (i.e., THN1-MlrC) towards linearized microcystins (MCs) and structural/physic-chemical properties of MIrC enzyme deserved urgent research, and heterologous expression (HE) optimization for MIrC is yet to be solved. This study achieved HE of THN1-MIrC by rapid -efficiently constructing HE system, and revealed that THN1-MIrC can degrade linearized MC-LR and linearized MC-RR to produce Adda, providing direct evidence for catalytic function of THN-MIrC and its ecological implication in MC-detoxification. Consequently, to maximize THN1-MIrC expression for production and application, induction conditions for HE of THN1-MIrC was optimized as at 0.1 mM of isopropyl-(3-D-thiogalactoside and 30 C for 8 h, which could be widely applicable for heterologous production of other MIrC homologs. Using bioinformatics and site-mutation experiment, THN1-MIrC was evaluated as a cytoplasm-locating hydrophilic protein with theoretical isoelectric point of 5.57, and contained six verified active sites G1u39, His133, Asp167,His169,His191 and Asp332 in two domains of its 3D structure, among which Glu39, His133 and Asp332 were newly-discovered ones here. The G1u39, His133, Asp167, His169 and HI..-s191 gathered more closely in 3D structure than in amino acid sequence, while they and Asp332 surrounded protein center to constitute a potential active pocket for mediating linearized MCs degradation. Due to MIrC sequence homology and conservative active sites, structural/physic-chemical characteristics of THN1-MIrC presented in this study provided a helpful reference for other MIrC homologs of diverse bacteria. This study shed novel insights for functional-structural relationships of THN1-MIrC during MC-biodegradation, and was crucial for research and practical applications in MC-decontamination. (C) 2020 Elsevier Ltd. All rights reserved.