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Biochemistry. 2018 Feb 13;57(6):963-977. doi: 10.1021/acs.biochem.7b01137. Epub 2018 Jan 26.

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

Author information

1
Department of Molecular Physiology and Biological Physics, University of Virginia , 1340 Jefferson Park Avenue, Charlottesville, Virginia 22908, United States.
2
Laboratory for Structural and Biochemical Research, Biological and Chemical Research Centre, Department of Chemistry, University of Warsaw , 101 Zwirki i Wigury, 02-089 Warsaw, Poland.
3
Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw , 93 Zwirki i Wigury, 02-089 Warsaw, Poland.
4
Department of Chemistry, University of Warsaw , 1 Ludwika Pasteura, 02-093 Warsaw, Poland.

Abstract

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.

PMID:
29309127
DOI:
10.1021/acs.biochem.7b01137
[Indexed for MEDLINE]

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