1HUV

CRYSTAL STRUCTURE OF A SOLUBLE MUTANT OF THE MEMBRANE-ASSOCIATED (S)-MANDELATE DEHYDROGENASE FROM PSEUDOMONAS PUTIDA AT 2.15A RESOLUTION

Summary for 1HUV

• Entry DOI: 10.2210/pdb1huv/pdb
• Descriptor: L(+)-MANDELATE DEHYDROGENASE, SULFATE ION, FLAVIN MONONUCLEOTIDE, ... (5 entities in total)
• Functional Keywords: tim barrel, oxidoreductase
• Biological source: Pseudomonas putida; More
• Cellular location: Membrane: P20932
• Total number of polymer chains: 1
• Total formula weight: 42879.92

Authors

Mathews, F.S.,Sukumar, N. (deposition date: 2001-01-04, release date: 2001-09-19, Last modification date: 2023-08-09)

Primary citation

Sukumar, N.,Xu, Y.,Gatti, D.L.,Mitra, B.,Mathews, F.S.
Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase.
Biochemistry, 40:9870-9878, 2001

PubMed 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.

PubMed: 11502180
DOI: 10.1021/bi010938k

Experimental method

X-RAY DIFFRACTION (2.15 Å)