Functional and structural analyses for MlrC enzyme of Novosphingobium sp. THN1 in microcystin-biodegradation: Involving optimized heterologous expression, bioinformatics and site-directed mutagenesis
2020
Wang, Ruiping | Li, Jieming | Li, Ji
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.
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