Structural basis of microcystinase activity for biodegrading microcystin-LR.

Microcystinase (MlrA) catalyzes the first and most important biodegradation step of hepatotoxic microcystin-LR (MC-LR) produced and released from cyanobacterial cells, and the underlying catalytic mechanism is not completely understood yet. MlrA was postulated previously to be a metalloprotease with an active site of HAIHNE, a variant of the common metal-binding motif of HEXXH. Through comparison with representative modes in HEXXH-containing metalloproteases, molecular dynamics simulation, homology modeling, and docking, the active sites of MlrA involved in the MC-LR biodegradation by Sphingomonas sp. USTB-05 were predicted. Site-directed mutants of MlrA were constructed for verification then. The results show that MlrA is likely not a metalloprotease, but a glutamate protease belonging to type II CAAX prenyl endopeptidases. Combined with the biodegradation of MC-LR by MlrA and its mutants, a complete enzymatic mechanism for MC-LR biodegradation by MlrA is proposed: Glu and His activate a water molecule facilitating a nucleophilic attack on the Adda-Arg peptide bond of MC-LR; Trp and Trp contact the carboxylate side chain of Gluand, by raising its pKa potentially, accelerate the reaction rates; His and Asn (located in the previous postulated active center of HAIHNE) function as an oxyanion hole to stabilize the transition states. This study reveals the enzymatic mechanism of MlrA for catalyzing MC-LR in both the representative modes and the experiments of site-directed mutagenesis.