Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase

Biochemistry. 2001 Aug 21;40(33):9870-8. doi: 10.1021/bi010938k.

Abstract

The structure of an active mutant of (S)-mandelate dehydrogenase (MDH-GOX2) from Pseudomonas putida has been determined at 2.15 A resolution. The membrane-associated flavoenzyme (S)-mandelate dehydrogenase (MDH) catalyzes the oxidation of (S)-mandelate to give a flavin hydroquinone intermediate which is subsequently reoxidized by an organic oxidant residing in the membrane. The enzyme was rendered soluble by replacing its 39-residue membrane-binding peptide segment with a corresponding 20-residue segment from its soluble homologue, glycolate oxidase (GOX). Because of their amphipathic nature and peculiar solubilization properties, membrane proteins are notoriously difficult to crystallize, yet represent a large fraction of the proteins encoded by genomes currently being deciphered. Here we present the first report of such a structure in which an internal membrane-binding segment has been replaced, leading to successful crystallization of the fully active enzyme in the absence of detergents. This approach may have general application to other membrane-bound proteins. The overall fold of the molecule is that of a TIM barrel, and it forms a tight tetramer within the crystal lattice that has circular 4-fold symmetry. The structure of MDH-GOX2 reveals how this molecule can interact with a membrane, although it is limited by the absence of a membrane-binding segment. MDH-GOX2 and GOX adopt similar conformations, yet they retain features characteristic of membrane and globular proteins, respectively. MDH-GOX2 has a distinctly electropositive surface capable of interacting with the membrane, while the opposite surface is largely electronegative. GOX shows no such pattern. MDH appears to form a new class of monotopic integral membrane protein that interacts with the membrane through coplanar electrostatic binding surfaces and hydrophobic interactions, thus combining features of both the prostaglandin synthase/squaline-hopine cyclase and the C-2 coagulation factor domain classes of membrane proteins.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics*
  • Amino Acid Sequence
  • Binding Sites
  • Cell Membrane / metabolism
  • Conserved Sequence
  • Crystallography, X-Ray
  • Flavins / chemistry
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Oxygen / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Pseudomonas putida / enzymology
  • Recombinant Fusion Proteins
  • Sequence Homology, Amino Acid

Substances

  • Flavins
  • Recombinant Fusion Proteins
  • Alcohol Oxidoreductases
  • glycollate oxidase
  • L-mandelate dehydrogenase
  • Oxygen

Associated data

  • PDB/1HUV