QM/MM study of the [4Fe-4S]-dependent (R)-2-hydroxyisocaproyl-CoA dehydratase: Dehydration via a redox pathway with an a-carbonyl radical intermediate

The dehydration of proteinogenic amino acids is an essential fermentation process mediated by the (R)-2-hydroxyacyl-CoA dehydratase enzyme family. (R)-2-hydroxyisocaproyl-CoA dehydratase catalyzes the dehydration of (R)-2-hydroxyisocaproyl-CoA to harvest an E- product in the fermentation of L-leucine. In this work, QM/MM calculations were performed to elucidate the mechanism and stereoselectivity of this enzyme. Six possible antiferromagnetically coupled spin states (aabb, abab, abba, bbaa, baba, baab) were considered for the [4Fe-4S] cluster. First, the binding mode of the substrate to the reduced [4Fe-4S]' cluster switches from carbonyl oxygen coordination to a-hydroxy group coordination. This ligand exchange is coupled with the formation of a hydrogen bond between the substrate carbonyl oxy-gen and a second-shell serine residue, leading to an electron transfer from the [4Fe-4S]' cluster to the substrate and the formation of a ketyl radical anion intermediate. This is followed by the heterolytic cleavage of the substrate C-OH bond, which is coupled with a spin-center shift process, generating a Fe-coordinated hydroxide and an a-carbonyl radical intermediate. Subsequently, the Fe-coordinated hydroxide abstracts the b-proton from the substrate, generating the experimentally identified allylic ketyl radical product. The syn-elimination to afford an E-product is kinetically more favorable than the anti-elimination (Z-isomer). The results highlighted the importance of using a redox pathway to activate inert substrates for dehydration, which should be helpful for the mechanistic understanding of related enzymes in the [4Fe-4S] cluster-dependent dehydratase family. (c) 2023 Published by Elsevier Inc.