İstanbul INTERNATIONAL MODERN SCIENTIFIC RESEARCH CONGRESS, İstanbul, Türkiye, 4 - 05 Haziran 2021, cilt.1, sa.1, ss.4-5
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 1.2.1.2, 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.