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Ordu E., Gök Ş.

İstanbul INTERNATIONAL MODERN SCIENTIFIC RESEARCH CONGRESS, İstanbul, Turkey, 4 - 05 June 2021, vol.1, no.1, pp.4-5

  • Publication Type: Conference Paper / Summary Text
  • Volume: 1
  • City: İstanbul
  • Country: Turkey
  • Page Numbers: pp.4-5
  • Yıldız Technical University Affiliated: Yes


ENGINEERING OF SELECTED CYSTEINE RESIDUES OF NAD+ DEPENDENT FORMATE DEHYDROGENASE TO INVESTIGATE THEIR ROLE IN PROTEIN STABILITY ABSTRACT The most important feature demanded in industrial enzymes is their stablity under the harsh conditions of industrial synthesis. Therefore, comprehensive elucidation of protein stability is critical to design new enzymes that have improved activity or stability. NAD+ -dependent formate dehydrogenases (EC, FDH) are industrially important enzymes in the regeneration of NAD(P)H which is an expensive coenzyme used in the synthesis of chiral molecules requested to be optically pure, and the reduction of CO2 to product formate which is a stabilized form of hydrogen fuel, because they catalyze the oxidation of formate into CO2 concomitant with the reduction of NAD+ to NADH, while it catalyzes reduction of the CO2 into formate under appropriate conditions. Plant FDHs also play a crucial role in the maintenance of a reducing environment to combat oxidative stress in plants. Recombinant FDH from Gossypium hirsutum contains 5 cysteine amino acids in the C22, C51, C185, C218, C267 residues. It has been shown that the oxidation of Cys residues to the sulfoxide forms reduces stability and activity depending on their position in the protein structure. In this study, it is aimed to investigate the effect of free cysteine residues of the GhFDH enzyme on the stability of enzyme. Site-directed mutagenesis method has been applied for the substitution of Cys51 and Cys267 residues to Leucine based on the homology modelling results obtained by using crystal structure of FDH from Arabidopsis thalinana as a template. Sequence analysis confirmed that targeted changes were achieved successfully. Characterization studies to clarify the activity and stability of mutant enzymes are in progress. Preliminary results showed that Cys51Leu is more tolerant to oksidative stress that that of recominant native GhFDH. Keywords: NAD+ Dependent Format Dehydrogenase, Cysteine Residues, Oxidative Stability This work has been supported by Yildiz Technical University Scientific Research Projects Coordination Unit under project number FYL-2021-4224.