Computational study of the covalent bonding of microcystins to cysteine residues – a reaction involved in the inhibition of the PPP family of protein phosphatases

Abstract

Microcystins (MCs) are cyclic peptides, produced by cyanobacteria, that are hepatotoxic to mammals. The toxicity mechanism involves the potent inhibition of protein phosphatases, as the toxins bind the catalytic subunits of five enzymes of the phosphoprotein phosphatase (PPP) family of serine ⁄ threonine-specific phosphatases: Ppp1 (aka PP1), Ppp2 (aka PP2A), Ppp4, Ppp5 and Ppp6. The interaction with the proteins includes the formation of a covalent bond with a cysteine residue. Although this reaction seems to be accessory for the inhibition of PPP enzymes, it has been suggested to play an important part in the biological role of MCs and furthermore is involved in their nonenzymatic conjugation to glutathione. In this study, the molecular interaction of microcystins with their targeted PPP catalytic subunits is reviewed, including the relevance of the covalent bond for overall inhibition. The chemical reaction that leads to the formation of the covalent bond was evaluated in silico, both thermodynamically and kinetically, using quantum mechanical-based methods. As a result, it was confirmed to be a Michael-type addition, with simultaneous abstraction of the thiol hydrogen by a water molecule, transfer of hydrogen from the water to the a,b-unsaturated carbonyl group of the microcystin and addition of the sulfur to the b-carbon of the microcystin moiety. The calculated kinetics are in agreement with previous experimental results that had indicated the reaction to occur in a second step after a fast noncovalent interaction that inhibited the enzymes per se.

Microcystin-Bound Protein Patterns in Different Cultures of Microcystis aeruginosa and Field Samples

“…They also suggested that certain other conditions, including temperature, free/protein-bound MC concentration and pKa values of the thiols present could influence the equilibrium between conjugation and deconjugation. The reversibility could be possible, as the conjugation reaction between an α,β-unsaturated carbonyl of MC and free thiol of the cysteine of specific biomolecules/proteins is a Michael-type addition reaction [39] and several studies have also discovered that some Michael additions of oxygen, nitrogen and sulfur nucleophiles to α,β-unsaturated carbonyls were reversible [40,41,42,43]. Miles et al [38] pointed out that the environmental and toxicological consequences of the reversibility in aquatic organisms need further investigation.…”

Reactivity of Biliatresone, a Natural Biliary Toxin, with Glutathione, Histamine, and Amino Acids

“…23 Reduction of a microcystin to its dihydromicrocystin abolished the ability of the toxin to form covalent bonds with the enzyme, suggesting the importance of the Michael addition of the α -methylene and cysteine residue in protein activation and toxicity. 36,37 Understanding the reactivity of biliatresone may enable us to identify similar molecules with relevance to human biliary atresia.…”

Seven new microcystin variants discovered from a native Microcystis aeruginosa strain – unambiguous assignment of product ions by tandem mass spectrometry

“…[7] Studying MCs is important because of their harmful impact on both ecological systems and humans. MCs specifically inhibit protein serine/threonine phosphatases using the Adda and Mdha residues, [10,11] leading to increased phosphorylation in human cells and intrahepatic haemorrhage, haemodynamic shock, heart failure, and death. [4] It has also been reported that MCs inactivate tumor suppressor genes [12] and form nodules associated with morphologic changes in hepatocytes.…”