Crystal structural analysis and characterization for MlrC enzyme of Sphingomonas sp. ACM-3962 involved in linearized microcystin degradation.

Microcystinase C (MlrC), one key hydrolase of the microcystinase family, plays an important role in linearized microsystin (L-MC) degradation. However, the three-dimensional structure and structural features of MlrC are still unclear. This study obtained high specific activity and high purity of MlrC by heterologous expression, and revealed that MlrC derived from Sphingomonas sp. ACM-3962 (ACM-MlrC) can degrade linearized products of MC-LR, MC-RR and MC-YR to product 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda), indicating the degradation function and significance in MC-detoxification. More importantly, this study reported the crystal structure of ACM-MlrC at 2.6 Å resolution for the first time, which provides a basis for further understanding the structural characteristics and functions of MlrC. MlrC had a dual-domain feature, namely N and C terminal domain respectively. The N-terminal domain contained a Glutamate-Aspartate-Histidine-Histidine catalytic quadruplex coordinated with zinc ion in each monomer. The importance of zinc ions and their coordinated residues was analyzed by dialysis and site-directed mutagenesis methods. Moreover, the important influence of the N/C-terminal flexible regions of ACM-MlrC was also analyzed by sequence truncation, and then the higher yield and total activity of variants were obtained, which was beneficial to study the better function and application of MlrC.