Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.
Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.
Najmudin
Shabir
S
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England.
Andersen
Jens T
JT
Patkar
Shamkant A
SA
Borchert
Torben V
TV
Crout
David H G
DH
Fülöp
Vilmos
V
D001412:Bacillus subtilis / Q000201:enzymology; D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray
D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase