Crystallization of and X-ray investigations on glucose dehydrogenase from Bacillus megaterium

The tetrameric glucose dehydrogenase from Bacillus megaterium M1286 belongs to the 'short' family of dehydrogenases with 262 amino acids per subunit (Mr approximately 30,000), and does not require Zn2+ for enzymatic action. It was crystallized as complex with its coenzyme NAD from a 1-2% protein solution by the batch method using ammonium sulfate as precipitant at pH 6.5. Crystals appeared within two days as clusters of large plates with maximum dimensions of 2 mm, which diffract X-rays to a resolution of at least 0.2 nm. The space group is orthorhombic P2(1)2(1)2(1), unit-cell dimensions are a = 15.03 nm, b = 10.42 nm and c = 6.74 nm. Assuming one molecule (approximately 120 kDa) per asymmetric unit the VM value is 0.0022 nm3/Da and the solvent content of the crystals is 45% based on a partial specific volume for the protein of 0.723 ml/g. The crystallization was further improved by using the microdialysis technique where instead of clusters, single crystals appeared within 7 days.