Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (ADHs): a soluble monomeric ADH from Pseudomonas putida HK5 (ADH-IIB) and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS).
Crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (ADH-IIB) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (ADH-GS) have been obtained. The ADH-IIB crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of PEG 8000, sodium citrate, HEPES buffer and 2-propanol. X-ray data were collected at 110 K to 1. 9 A resolution (R(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from Methylophilus methylotrophus W3A1) was obtained by molecular replacement. Preliminary refinement of this model (10.0-3.0 A resolution, R = 0.37, R(free) = 0.40) led to tentative identification of the two highest peaks in a native anomalous difference Fourier map as the Fe atom of the heme and a calcium ion interacting with the PQQ prosthetic group. The ADH-GS crystals are tetragonal, displaying six similar lattices, both primitive and centered, and were grown by the sitting-drop method after replacement of Triton X-100 by dodecylmaltoside or octaethylene glycol monododecyl ether in the presence of ammonium sulfate and sodium acetate buffer, with and without PEG 3500 and calcium ion. The best diffraction is obtained at 110 K where the resolution extends to about 4 A in the a and b directions and about 3 A in the c direction.
Chen
Z
Z
Department of Biochemistry, Washington University, St Louis, MO, USA.
Baruch
P
P
Mathews
F S
FS
Matsushita
K
K
Yamashita
T
T
Toyama
H
H
Adachi
O
O
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D003580:Cytochromes / Q000737:chemistry; D003902:Detergents; D020576:Gluconobacter; D045542:PQQ Cofactor; D011092:Polyethylene Glycols; D016958:Pseudomonas putida; D015363:Quinolones / Q000737:chemistry; D011809:Quinones / Q000737:chemistry
D003580:Cytochromes; D003902:Detergents; D015363:Quinolones; D011809:Quinones; D011092:Polyethylene Glycols; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)