Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 59; no. 6; pp. 1073 - 1075
• Main Authors: Najmudin, Shabir, Andersen, Jens T., Patkar, Shamkant A., Borchert, Torben V., Crout, David H. G., Fülöp, Vilmos
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England Munksgaard International Publishers 01-06-2003
• Subjects: acetolactate decarboxylase; Bacillus subtilis - enzymology; bifunctional enzymes; brewing; Carboxy-Lyases - biosynthesis; Carboxy-Lyases - chemistry; Carboxy-Lyases - isolation & purification; Cloning, Molecular; Crystallization; Crystallography, X-Ray
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444903006978

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 α‐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 P31,221, with unit‐cell parameters a = 47.0, c = 198.9 Å. A complete data set was collected to 2 Å from a single native crystal using synchrotron radiation.