Biotransformation is recognized as a potential pathway to regulate the environmental risk of microcystins (MCs). To explore the regulation effectiveness and mechanism of the biotransformation pathway, six typical MCLR-biotransformation products (MCLR-BTPs) were prepared, and their inhibition effects on protein phosphatase 2A (PP2A) were evaluated. The inhibition effects of the MCLR-BTPs generally decreased with the increase in biothiol molecular weights and polarity, indicating that biotransformation was an effective pathway through which to regulate MCLR toxicity. To further explore the regulation mechanism, the key interaction processes between the MCLR/MCLR-BTPs and the PP2A were explored by homology modeling and molecular docking. The introduced biothiols blocked the covalent binding of Mdha7 to Cys269 but strengthened the hydrogen bond “Mdha7”→Arg268. The changed “Mdha7” intervened the combination of MCLR-BTPs to PP2A by weakening the hydrogen bonds Arg4←Arg214, Arg4→Pro213, Adda5←His118, and Ala1←Arg268, and the ionic bond Glu6-Mn12+. The weakening combination of the MCLR-BTPs to PP2A further attenuated the interactions between the conserved domain and the Mn2+ ions (including the ionic bonds Asp57-Mn12+ and Asp85-Mn12+ and the metal bonds Asp57-Mn12+ and Asn117-Mn12+) and increased the exposure of the Mn2+ ions. Meanwhile, the weakened hydrogen bond Arg4←Arg214 facilitated the combination of the phosphate group to Arg214 (with increased exposure). In this way, the catalytic activity of the PP2A was restored.

The secondary contamination of microcystin disinfection by-products (MC-DBPs) is of concern due to the residual structure similar to their original toxin. Based on identification and preparation, the potential inhibition effect of typical MCLR-DBPs (associated with the oxidation of Adda5) on PP2A was confirmed in the sequence of MCLR > P1 > P4 > P3 ≈ P2 > P7 ≈ P6 ≈ P5 > P8. To elucidate the molecular mechanism underlying the inhibition effect, the interaction models for typical MCLR-DBPs and PP2A were constructed using a modeling-based-on-ligand-similarity approach, and the candidate interaction parameters between typical MCLR-DBPs and PP2A were obtained by molecular docking. By analyzing the correlation between inhibition data and candidate interaction parameters, the key interaction parameters were filtered as hydrogen bonds “Adda5”←Asn117, “Adda5”←His118, MeAsp3←Arg89, Arg4←Arg214, Arg4→Pro213; ionic bonds Glu6-Arg89, Asp85-Mn12+, Asp57-Mn22+; and metal bonds Glu6-Mn12+, Glu6-Mn22+. With the gradual intensification of chlorination, Adda5 was destroyed to varying degrees. The key interactions changed correspondingly, resulting in the discrepant inhibition effects of typical MCLR-DBPs on PP2A.

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Microcystins (MCs) exhibit diversified inhibition effects on protein phosphatases (PPs) due to their structural differences. To fully evaluate the potential mechanism for the discrepant inhibition effects, the five most frequent MCs with varying residues at position Z4 were selected as the tested toxins. Their inhibition sequence on PP2A was detected as follows: MCLR > MCLW > MCLA > MCLF > MCLY. Combined with homology modeling and molecular docking technology, the major interaction parameters between the MCs and PP2A were obtained. The correlation analysis for the major interaction parameters and inhibition effects showed that the hydrophobicity of Z4 had an important influence on the interaction of the MCs to PP2A. The introduction of hydrophobic Z4 directly weakened hydrogen bonds Z4→Pro213 and Z4←Arg214, indirectly weakened hydrogen bonds Adda5←Asn117, Glu6←Arg89, and MeAsp3←Arg89, but indirectly enhanced ionic bonds Glu6←Arg89, Glu6-Mn12+, and Glu6-Mn22+. In this way, the combination of the MCs with PP2A was blocked, and thus, the interactions between PP2A and the Mn2+ ions (in the catalytic center) were further affected; metal bonds Asp85-Mn12+ and Asp85-Mn22+ were weakened, while metal bond His241-Mn12+ was enhanced. As a result, the interactions in the catalytic center were inhibited to varying degrees, resulting in the reduced toxicity of MCs.

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