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 MlrC from Novosphingobium sp. THN1 (i.e., THN1-MlrC) towards linearized microcystins (MCs) and structural/physic-chemical properties of MlrC enzyme deserved urgent research, and heterologous expression (HE) optimization for MlrC is yet to be solved. This study achieved HE of THN1-MlrC by rapid-efficiently constructing HE system, and revealed that THN1-MlrC can degrade linearized MC-LR and linearized MC-RR to produce Adda, providing direct evidence for catalytic function of THN-MlrC and its ecological implication in MC-detoxification. Consequently, to maximize THN1-MlrC expression for production and application, induction conditions for HE of THN1-MlrC was optimized as at 0.1 mM of isopropyl-β-D-thiogalactoside and 30 °C for 8 h, which could be widely applicable for heterologous production of other MlrC homologs. Using bioinformatics and site-mutation experiment, THN1-MlrC was evaluated as a cytoplasm-locating hydrophilic protein with theoretical isoelectric point of 5.57, and contained six verified active sites Glu39, 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 Glu39, His133, Asp167, His169 and His191 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 MlrC sequence homology and conservative active sites, structural/physic-chemical characteristics of THN1-MlrC presented in this study provided a helpful reference for other MlrC homologs of diverse bacteria. This study shed novel insights for functional-structural relationships of THN1-MlrC during MC-biodegradation, and was crucial for research and practical applications in MC-decontamination.