Kratom medium-chain dehydrogenase/reductase enzymes exhibit broad substrate and cofactor promiscuity.

Medium-chain alcohol dehydrogenases/reductases (MDRs) are crucial to the biosynthesis of plant monoterpene indole alkaloids (MIAs), yet studies have focused solely on their catalytic activities with strictosidine aglycone derivatives and NADPH as the redox cofactor. Here, we combined computational and experimental approaches to characterize the substrate and cofactor promiscuity of five MDRs recently identified in the medicinal plant kratom, including MsMDR4, MsMDR11, MsTHAS (tetrahydroalstonine synthase), MsDCS1 (dihydrocorynantheine synthase), and MsHYS (heteroyohimbine synthase). In vitro and yeast-based biochemical analyses showed that MsMDR4, MsMDR11, and MsTHAS catalyze the aldehyde reduction for cinnamaldehyde and 8-oxogeraniol, while MsMDR11 and MsTHAS also catalyze the alcohol oxidation for cinnamyl alcohol and 8-hydroxygeraniol. In particular, MsMDR11 exhibits 8-hydroxygeraniol oxidoreductase activity, producing 8-oxogeranial, an upstream intermediate in the strictosidine pathway. Additionally, all five enzymes can accept NADH/NAD + as redox cofactors, though with decreased productivity. Swapping the reducing cofactor from NADPH to NADH further alters the distribution of MIAs produced by MsDCS1 and MsHYS. Together, kratom MDRs exhibit great plasticity and potential for future enzyme engineering. One-sentence summary Computational and biochemical analyses revealed that previously identified kratom medium-chain dehydrogenase/reductases exhibit broad substrate and cofactor promiscuity.