Crystallization and preliminary X-ray crystallographic studies on the class II cholesterol oxidase from Burkholderia cepacia containing bound flavin.
Burkholderia cepacia cholesterol oxidase (ChoS) is a 58.7 kDa molecular-weight flavoenzyme which has been categorized as a 3beta-hydroxysteroid oxidase converting the 3beta-hydroxyl group of a range of hydroxysteroids to the corresponding ketone. Analysis of enzymes with this activity has shown that two classes of cholesterol oxidase can be defined. Enzymes belonging to class I contain non-covalently bound FAD, whereas the class II enzymes contain FAD covalently bound to an active-site histidine. Despite catalysing the same chemical reaction, the class I and class II enzymes show no sequence similarity and have a different molecular architecture. Crystals of a recombinant class II enzyme from B. cepacia have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals belong to space group P3(1)21, with unit-cell parameters a = b = 119.6, c = 101.1 A, and have one subunit in the asymmetric unit. These crystals diffract to at least 2.0 A resolution at the Daresbury SRS and are suitable for a full structure determination. Ultimately, analysis of the structure of B. cepacia ChoS may allow the characteristics and structural features which contribute to its suitability as a diagnostic reagent for the detection of cholesterol and unresolved mechanistic features of the class II enzymes to be understood.
Aunpad
Ratchaneewan
R
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, England.
Muench
Stephen P
SP
Baker
Patrick J
PJ
Sedelnikova
Svetlana
S
Panbangred
Watanalai
W
Doukyu
Noriyuki
N
Aono
Rikizo
R
Rice
David W
DW
D016956:Burkholderia cepacia / Q000201:enzymology*; D002789:Cholesterol Oxidase / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D005415:Flavins / Q000737:chemistry*; D011487:Protein Conformation
D005415:Flavins; D002789:Cholesterol Oxidase