Crystallization behaviour of glyceraldehyde dehydrogenase from Thermoplasma acidophilum

Iu. Iermak^1,2, O. Degtjarik^1,3, F. Steffler⁴, V. Sieber⁵ and I. Kuta Smatanova^1,2

¹Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 37005 Ceske Budejovice, Czech Republic,

²Center of Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Zamek 136, 37333 Nove Hrady, Czech Republic,

³Weizmann Institute of Science, Department of Structural Biology, 76100 Rehovot, Israel

⁴Bio, Electro- and Chemocatalysis BioCat, Straubing Branch of the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Schulgasse 11a, 94315 Straubing, Germany,

⁵Chemistry of Biogenic Resources, Straubing Centre of Science, Technische Universitat Munchen, Schulgasse 16, 94315 Straubing, Germany.

julia.ermak90@gmail.com

The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a microbial enzyme that catalyses the oxidation of Dglyceraldehyde to D-glycerate in the artificial enzyme cascade designed for the conversion of glucose to the organic solvents isobutanol and ethanol [1, 2]. Various mutants of TaAlDH were constructed by random approach followed by site-directed and saturation mutagenesis in order to improve enzyme properties essential for its functioning within the cascade [3].

For further improvement of TaAlDH, it will be more effective to modify the enzyme at target positions via rational design. Since available TaAlDH protein models are poor (homology < 40%), a TaAlDH crystal structure would allow for distinct enzyme modifications with predictable impact on activity and stability.

Variously shaped crystals grew within one-two weeks after initial screening in 30 diverse conditions for TaAlDH wild type and 24 different conditions for TaAlDH F34M+S405N mutant. In order to obtain the best quality crystals optimization was carried out considering following parameters: (a) already known diffraction quality of crystals; (b) size and shape of crystals (big single crystals with sharp edges preferred); (c) visually different crystal forms (to check as many as possible different variants of protein molecules packing inside the crystal). Optimization, including variation of pH, protein and precipitant concentrations and ratios, resulted in diffracting crystals only from one condition for TaAlDHwt and two conditions for TaAlDH F34M+S405N. Crystals from other conditions were poorly reproducible and diffracted only to 8-10 Å resolution even after microseeding procedure.

Crystals of TaAlDHwt belong to monoclinic P12₁1 space group with 8 molecules per asymmetric unit and diffracted to the resolution of 1.95 Å. TaAlDH F34M+S405N crystallizes in two different space groups: triclinic P1 with 16 molecules per asymmetric unit and monoclinic C121 with 4 molecules per asymmetric unit. These crystals diffracted to the resolutions of 2.14 and 2.10 Å for P1 and C121, respectively [4].

1. Guterl, J.-K., Garbe, D., Carsten, J., Steffler, F., Sommer, B., Reiße, S., Philipp, A., Haack, M., Ruhmann, B., Koltermann, A., Kettling, U., Bruck, T. & Sieber, V. ChemSusChem, 5, (2012), 2165–2172.

2. Steffler, F. & Sieber, V. PLOS ONE 8, (2013), e70592.

3. Steffler, F., Guterl, J.-K. & Sieber, V. Enzyme Microb. Tech. 53, (2013), 307-314.

4. Iermak, I., Degtjarik, O., Steffler, F., Sieber, V., & Kuta Smatanova, I. Acta Cryst. F, 71(12), (2015), 1475-1480.

We would like to express our thanks to Dr. Jörg Carsten and Anja Schmidt (Chemistry of Biogenic Resources, Straubing Centre of Science, Technische Universität München) for technical assistance and MX user support team of BESSY II, Helmholtz-Zentrum Berlin for their help during the data collection. Support from the University of South Bohemia in Ceske Budejovice and AS CR is appreciated as well.