From NAD⁺ to Nickel Pincer Complex: A Significant Cofactor Evolution Presented by Lactate Racemase

Lactate racemase (LarA), a new nickel enzyme discovered recently, catalyzes the racemization between d- and l-lactates with a novel nickel pincer cofactor (Ni-PTTMN) derived from nicotinic acid. In this study, by using DFT and a 200-atom active-site model, LarA is revealed to employ a modified proton-coupled hydride-transfer mechanism in which a hydride is transferred to a cofactor pyridine carbon from the substrate α-carbon along with proton transfer from the substrate hydroxy group to a histidine, and then moved back from the opposite side. Tyr294 and Lys298 provide significant acceleration effects by orientating substrates and stabilizing the negative charge developing at the substrate hydroxy oxygen. The barrier was determined to be 12.0 kcal mol⁻¹, which reveals enhanced racemase activity relative to the LarA reaction using NAD⁺-like cofactors. Compared with NAD⁺, Ni-PTTMN has a stronger hydride-addition reactivity in moderate and high environmental polarity and may fit perfectly the moderately polar active site of LarA.