Biotechnological production of chemical compounds is an environmentally more gentle process compared to their fabrication from natural fossil resources [1]. In terms of bio-production, cell-free processes are more effective than microbial production techniques since the enzymes can tolerate higher concentrations of final product than the cells. The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a part of an artificial cell-free enzyme cascade for production of isobutanol and ethanol from glucose. TaAlDH catalyzes the oxidation of Dglyceraldehyde to D-glycerate in this synthetic pathway [2]. Various mutants of TaAlDH were constructed by random approach followed by site-directed and saturation mutagenesis in order to improve the enzymes’ properties essential for its functioning within the cascade. Further optimization of TaAlDH requires structural information about the enzyme for which crystallization followed by X-ray diffraction analysis was employed [3].
Diffracting quality crystals of TaAlDH wild type were obtained after initial screening in condition H6 of the Morpheus screen (Molecular Dimensions Ltd., UK) followed by optimization, including variation of pH, protein and precipitant concentrations and ratios. Full data set was collected on the on the BL 14.1 operated by the Joint Berlin MXLaboratory at the BESSY II electron-storage ring (Berlin-Adlershof, Germany) at 1.95Å resolution. Crystals belong to monoclinic P21 space group with unit cell parameters of a = 95.29 Å, b = 152.35 Å, c = 149.90 Å, α = γ = 90.0°, β = 92.19°. Matthews coefficient of VM = 2.41 Å3 Da-1 suggests that the crystals contain 8 molecules per asymmetric unit with a solvent content of 48.95%.
The structure of TaAlDHwt was solved by molecular replacement using the coordinates of betaine-aldehyde dehydrogenase from Pseudoalteromonas atlantica T6c (sequence identity 38%, PDB ID 3K2W). The final model contains two tetramers in the asymmetric unit that are related by non-crystallographic symmetry with differences observed in regions participating in crystal contacts. The TaAlDHwt homotetramer consists of two homodimers that display a very tight connection through the formation of an extended beta-sheet between monomers of the dimer. The structure refinement of TaAlDHwt is in progress.