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Altering NAD(P)H cofactor specificity by structure‐guided modification of class II HMG‐CoA reductase

HMG‐CoA reductase (HMGR) is a central enzyme in metabolism and natural product biosynthesis, as it catalyzes the committed and rate‐determining step of the mevalonate pathway, which produces steroids and isoprenoids, the largest and most diverse class of natural products. Here, HMGR converts HMG‐CoA to mevalonate using two equivalents of a redox cofactor, either NADH or NADPH. Class I HMGRs are highly specific for NADPH, including human HMGR, the target of cholesterol‐lowering statin drugs. However, cofactor specificity of microbial class II HMGR varies widely, as some homologs exhibit a strong preference for either NADH or NADPH, while others homologs are less specific. We have recently identified a key structural feature termed the “cofactor helix” that may govern cofactor specificity in class II HMGR, but until now this helix’s role in conferring NAD(P)H preference has not been tested. In this work, we performed structure‐guided enzyme modification with the aim of altering the cofactor preferences of various class II HMGR homologs. Through kinetic and X‐ray crystallographic studies on these enzyme variants, we have found that the cofactor helix plays a critical but not exclusive role in controlling NAD(P)H preference, expanding our understanding of the structural determinants of cofactor specificity in class II HMGR.