Structural basis of microcystinase activity for biodegrading Microcystin-LR

Abstract 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 H 260 AIH 263 NE 265 , 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 172 and His 205 activate a water molecule facilitating a nucleophilic attack on the Adda-Arg peptide bond of MC-LR; Trp 176 and Trp 201 contact the carboxylate side chain of Glu 172 and, by raising its p K a potentially, accelerate the reaction rates; His 260 and Asn 264 (located in the previous postulated active center of H 260 AIH 263 NE 265 ) 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.