期刊
BIOCHEMISTRY
卷 57, 期 6, 页码 963-977出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.7b01137
关键词
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资金
- NIGMS [U54-GM094662, R01-GM117080]
- NIH Big Data to Knowledge (BD2K) [HG008424]
- NCN (Maestro) [UMO-2012/04/A/ST5/00609, UMO-2014/15/B/NZ1/03357]
- FNP (TEAM) grants
- UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research [DE-ACO2-06CH11357]
- Michigan Economic Development Corporation
- Michigan Technology Tri-Corridor [085P1000817]
The D-2-hydroxyacid dehydrogenase (2HADH) family illustrates a complex evolutionary history with multiple lateral gene transfers and gene duplications and losses. As a result, the exact functional annotation of individual members can be extrapolated to a very limited extent. Here, we revise the previous simplified view on the classification of the 2HADH family; specifically, we show that the previously delineated glyoxylate/hydroxypyruvate reductase (GHPR) subfamily consists of two evolutionary separated GHRA and GHRB subfamilies. We compare two representatives of these subfamilies from Sinorhizobium meliloti (SmGhrA and SmGhrB), employing a combination of biochemical, structural, and bioinformatics approaches. Our kinetic results show that both enzymes reduce several 2-ketocarboxylic acids with overlapping, but not equivalent, substrate preferences. SmGhrA and SmGhrB show highest activity with glyoxylate and hydroxypyruvate, respectively; in addition, only SmGhrB reduces 2-keto-D-gluconate, and only SmGhrA reduces pyruvate (with low efficiency). We present nine crystal structures of both enzymes in apo forms and in complexes with cofactors and substrates/substrate analogues. In particular, we determined a crystal structure of SmGhrB with 2-keto-D-gluconate, which is the biggest substrate cocrystallized with a 2HADH member. The structures reveal significant differences between SmGhrA and SmGhrB, both in the overall structure and within the substrate-binding pocket, offering insight into the molecular basis for the observed substrate preferences and subfamily differences. In addition, we provide an overview of all GHRA and GHRB structures complexed with a ligand in the active site.
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