New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases

Sci Rep. 2016 Feb 11;6:20629. doi: 10.1038/srep20629.

Abstract

Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Catalytic Domain
  • Crystallography, X-Ray
  • Enzyme Assays
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Glyceric Acids / chemistry
  • Glyceric Acids / metabolism
  • Glyoxylates / chemistry
  • Glyoxylates / metabolism
  • Hydroxypyruvate Reductase / chemistry*
  • Hydroxypyruvate Reductase / genetics
  • Hydroxypyruvate Reductase / metabolism
  • Kinetics
  • Models, Molecular
  • NAD / chemistry
  • NAD / metabolism
  • NADP / chemistry
  • NADP / metabolism
  • Protein Binding
  • Protein Stability
  • Pyrococcus / chemistry*
  • Pyrococcus / enzymology
  • Pyrococcus furiosus / chemistry*
  • Pyrococcus furiosus / enzymology
  • Pyrococcus horikoshii / chemistry*
  • Pyrococcus horikoshii / enzymology
  • Pyruvates / chemistry
  • Pyruvates / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Archaeal Proteins
  • Glyceric Acids
  • Glyoxylates
  • Pyruvates
  • Recombinant Proteins
  • NAD
  • NADP
  • glyceric acid
  • hydroxypyruvic acid
  • Alcohol Oxidoreductases
  • glyoxylate reductase
  • Hydroxypyruvate Reductase
  • glyoxylic acid