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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.
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