Abstract
Quinone reductases 1 and 2 (NQO1 and NQO2) are paralogous FAD-linked enzymes found in all amniotes. NQO1 and NQO2 have similar structures, and both catalyze the reduction of quinones and other electrophiles; however, the two enzymes differ in their cosubstrate preference. While NQO1 can use both redox couples NADH and NADPH, NQO2 is almost inactive with these cosubstrates and instead must use dihydronicotinamide riboside (NRH) and small synthetic cosubstrates such as N-benzyl-dihydronicotinamide (BNAH) for efficient catalysis. We used ancestral sequence reconstruction to investigate the catalytic properties of a predicted common ancestor and two additional ancestors from each of the evolutionary pathways to extant NQO1 and NQO2. In all cases, the small nicotinamide cosubstrates NRH and BNAH were good cosubstrates for the common ancestor and the enzymes along both the NQO1 and NQO2 lineages. In contrast, with NADH as cosubstrate, extant NQO1 evolved to a catalytic efficiency 100 times higher than the common ancestor, while NQO2 has evolved to a catalytic efficiency 3000 times lower than the common ancestor. The evolutionary analysis combined with site-directed mutagenesis revealed a potential site of interaction for the ADP portion of NAD(P)H in NQO1 that is altered in charge and structure in NQO2. The results indicate that while NQO1 evolved to have greater efficiency with NAD(P)H, befitting an enzymatic function in cells, NQO2 was under selective pressure to acquire extremely low catalytic efficiency with NAD(P)H. These divergent trajectories have implications for the functions of both enzymes.