Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase.
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.
Sukumar
N
N
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Xu
Y
Y
Gatti
D L
DL
Mitra
B
B
Mathews
F S
FS
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005415:Flavins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid
D005415:Flavins; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase; C021538:L-mandelate dehydrogenase; D010100:Oxygen