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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / classification
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism
  • Aldehyde Oxidoreductases / chemistry*
  • Aldehyde Oxidoreductases / classification
  • Aldehyde Oxidoreductases / genetics
  • Aldehyde Oxidoreductases / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / classification
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Crystallography, X-Ray
  • Hydroxypyruvate Reductase / chemistry*
  • Hydroxypyruvate Reductase / classification
  • Hydroxypyruvate Reductase / genetics
  • Hydroxypyruvate Reductase / metabolism
  • Kinetics
  • Models, Molecular
  • Phylogeny
  • Protein Conformation
  • Sinorhizobium meliloti / chemistry
  • Sinorhizobium meliloti / enzymology*
  • Sinorhizobium meliloti / genetics
  • Sinorhizobium meliloti / metabolism
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Alcohol Oxidoreductases
  • Hydroxypyruvate Reductase
  • D-2-hydroxyacid dehydrogenase
  • Aldehyde Oxidoreductases
  • glyoxylate dehydrogenase (acylating)