Crystallization of glyceraldehyde dehydrogenase from Thermoplasma acidophilum

I. Iermak¹, O. Degtjarik^1,2, Fabian Steffler³, Volker Sieber³, I. Kuta Smatanova^1,2

¹University of South Bohemia, Faculty of Science, Branišovska 31, CZ-37005 Česke Budejovice, Czech Republic

²Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Chemistry of Biogenic Resources, Straubing Centre of Science, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany

Due to the depletion of natural fossil resources, the development of environmentally gentle fuel and chemicals production processes is of key importance. One of such biotechnological approaches is a cell-free process called Synthetic Cascade Biomanufacturing.

The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a part of cell-free system for production of isobutanol and ethanol from glucose. It participates in oxidation of Dglyceraldehyde to D-glycerate in this synthetic pathway. Wild type of TaAlDH has high substrate selectivity and product tolerance but leaves place for optimization. A mutant of TaAlDH (F34M+Y399C+S405N), which has a 55.7-fold increased activity of the wild type enzyme, enhanced NAD⁺ acceptance and a slight solvent tolerance improvement, was obtained by a directed evolution approach.

For further optimization of TaAlDH functioning within the isobutanol and ethanol synthetic pathway structural information about the enzyme variant and its complex with the substrate is required. For this purpose crystallization of the enzyme by itself and in complex with cofactor or substrate, what will be followed by X-ray diffraction analysis, is used. Screening of crystallization conditions was performed by means of different crystallization screens using Gryphon crystallization robot (Art Robbins Instruments, USA).

Some hits were found in several conditions of Morpheus screen (Molecular Dimensions Ltd., UK). Results are well reproducible; crystals grow quite big (0.1 x 0.4 mm) but plane and multilayer. Preliminary diffraction analysis revealed bad quality of crystals, which diffracted only to 18-20 Å, so further optimization is necessary. For optimization diverse techniques were consequentially applied: variation of protein and precipitant concentrations, different pH of buffer, additive screen, addition of NAD⁺ and glyceraldehyde and microseeding using Seed Bead (Hampton Research, USA). Certain improvement of crystals was observed in microseeding experiments.