Crystallization and preliminary X-ray diffraction analysis of glyceraldehyde dehydrogenase from Thermoplasma acidophilum

 

I. Iermak1, O. Degtjarik1,2, F. Steffler3, V. Sieber3, I. Kuta Smatanova1,2

 

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

julia.ermak90@gmail.com

 

Synthetic Cascade Biomanufacturing is a cell free enzyme cascade developed for environmentally gentle chemicals production [1]. The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a part of such cell free system for production of isobutanol and ethanol from glucose. A mutant of TaAlDH carrying mutations F34M+Y399C+S405N was constructed in order to improve enzyme’s properties essential for its functioning within the cascade: substrate selectivity, activity, product tolerance and acceptance of NAD+ as a cofactor [2]. The aim of our research is solving the 3D structure of TaAlDH (F34M+Y399C+S405N) in order to explore the mutations influence on the enzyme’s function.

For this purpose protein crystallization with further X-ray diffraction analysis of obtained crystals has been used.

TaAlDH (F34M+Y399C+S405N) was successfully crystallized by means of different crystallization screens using the Gryphon crystallization robot (Art Robbins Instruments, USA). Two different forms of crystals appeared at room temperature: form A in condition G2 of Morpheus screen (Molecular Dimensions Ltd., UK) and form B in A1 of PEGs Suite (QIAGEN, Netherlands). Crystals from PEGs Suite were suitable for X-ray diffraction analysis without optimization of crystallization conditions (crystal form B). Crystals from Morpheus screen grew quite big (200 x 300 μm) but plane and multilayer. Therefore, the optimization of crystallization conditions by variation of protein and precipitant concentrations was provided. Certain improvement of crystals quality was observed after the optimization and the final size of crystals was about 100 x 100 x 500 μm (crystal form A). Full diffraction data sets were collected at the BESSY II electron-storage ring operated by the Joint Berlin MX­Laboratory (Berlin-Adlershof, Germany) up to the resolutions of 2.14 and 2.2 Å for crystal form A and B, respectively.

Further work on determination of TaAlDH mutant structure by molecular replacement is currently in progress.

1. F. Steffler, V. Sieber, PLoSONE, 8(7), (2013), e70592.

2. F. Steffler, J.-K. Guterl, V. Sieber, Enzyme and Microbial Technology, 53, (2013), pp. 307– 314.