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Cyanobacterial hepatotoxins which are produced and released by bloom-forming cyanobacteria may threaten the safety of drinking water for human beings and animals. Microcystinase (MlrA) catalyzes the first and most important step for biodegrading hepatotoxic microcystins (MCs) and nodularin (NOD), which hydrolyzes the cyclic hepatotoxin into the linear hepatotoxin as the first product. Here the recombinant MlrA of Sphingopyxis sp. USTB-05 for biodegrading hepatotoxins was firstly purified and its activity was also investigated. The purification process of MlrA was comprised of four steps, affinity purification of glutathione S-transferase (GST)-tagged MlrA, thrombin cleavage of GST tag, GST tag removal and thrombin removal with the benzamidine sepharose. The purified recombinant MlrA was found to have a strong ability to catalyze hepatotoxins, and initial microcystin-LR (MC-LR) of 19.9 mg/L and NOD of 25.2 mg/L were removed within 10 min and 12 h at the protein concentration of 60 mg/L, respectively. The results indicated that the enzyme MlrA with a high activity could be purified and obtained with the biotechnology process of expression and purification described here, which is very important in further studies on the enzyme structure and the enzymatic mechanism for biodegrading hepatotoxins. • The recombinant microcystinase MlrA originated from Sphingopyxis sp. USTB-05 was firstly purified. • The purified MlrA had a strong ability in the biodegrading hepatotoxins containing microcystin-LR and nodularin. [ABSTRACT FROM AUTHOR]

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 H260AIH263NE265, 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: Glu172 and His205 activate a water molecule facilitating a nucleophilic attack on the Adda-Arg peptide bond of MC-LR; Trp176 and Trp201 contact the carboxylate side chain of Glu172and, by raising its p K a potentially, accelerate the reaction rates; His260 and Asn264 (located in the previous postulated active center of H260AIH263NE265) 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. Image 1 • Microcystinase (MlrA) was predicted belonging to type II CAAX prenyl endopeptidases. • E172, H205, H260, and N264 are critical residues responsible for MlrA activity. • Homology modeling of MlrA was verified positively by site-directed mutagenesis. [ABSTRACT FROM AUTHOR]