Structural, Biochemical, and Evolutionary Characterizations of Glyoxylate/Hydroxypyruvate Reductases Show Their Division into Two Distinct Subfamilies

heD-2-hydroxyacid dehydrogenase (2HADH)family illustrates a complex evolutionary history with multiplelateral gene transfers and gene duplications and losses. As aresult, the exact functional annotation of individual memberscan be extrapolated to a very limited extent. Here, we revisethe previous simplified view on the classification of the2HADH family; specifically, we show that the previouslydelineated glyoxylate/hydroxypyruvate reductase (GHPR)subfamily consists of two evolutionary separated GHRA and GHRB subfamilies. We compare two representatives of thesesubfamilies fromSinorhizobium meliloti(SmGhrA andSmGhrB), employing a combination of biochemical, structural, andbioinformatics approaches. Our kinetic results show that both enzymes reduce several 2-ketocarboxylic acids with overlapping,but not equivalent, substrate preferences.SmGhrA andSmGhrB show highest activity with glyoxylate and hydroxypyruvate,respectively; in addition, onlySmGhrB reduces 2-keto-D-gluconate, and onlySmGhrA reduces pyruvate (with low efficiency). Wepresent nine crystal structures of both enzymes inapoforms and in complexes with cofactors and substrates/substrate analogues.In particular, we determined a crystal structure ofSmGhrB with 2-keto-D-gluconate, which is the biggest substrate cocrystallizedwith a 2HADH member. The structures reveal significant differences betweenSmGhrA andSmGhrB, both in the overallstructure and within the substrate-binding pocket, offering insight into the molecular basis for the observed substrate preferencesand subfamily differences. In addition, we provide an overview of all GHRA and GHRB structures complexed with a ligand in theactive site.