What are Oxidoreductases? Oxidoreductases are a class of enzymes that catalyze oxidoreduction reactions Oxidoreductases catalyze the transfer of electrons from one molecule the oxidant to another molecule the reductant. Oxidorecuctases can be oxidases or dehydrogenases.
Oxidases are enzymes involved when molecular oxygen acts as an acceptor of hydrogen or electrons. Other oxidoreductases include peroxidases, hydroxylases, oxygenases, and reductases. Oppermann, U. Steroid Biochem. Toxicology , 71— Chang, N. Shafqat, J. USA 93, — USA 91, — Schwartz, M. Thatcher, D. Krook, M. Biochemistry 29, — Biochemistry 34, — Persson, B. Nordling, E. Klimacek, M. Rossmann, M. In: The Enzymes, 3rd edn. II, pp. Adams, M. Nature , — USA 70, — Buehner, M. Kallberg, Y.
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Determination of optimum buffering conditions for DHK To determine the optimum pH and the buffer concentration for maximum enzyme activity we considered three different buffers based on their buffering ability at certain pH range namely, mM sodium citrate pH 4.
Determination of reaction mechanism for DHK In order to understand the reaction mechanism of DHK, the activity of the enzyme was monitored spectrophotometrically at nm by varying both the substrate and cofactor concentration under standard reaction conditions. Determination of kinetic constants For enzyme kinetics, thermal shift and activity assay experiments mM Sodium Phosphate Buffer, mM NaCl was used for performing initial velocity and activity studies were monitored by NADPH utilization.
Fingerprint based clustering and binding mode determination Total compounds were considered for MACCS [ 26 , 27 ] based clustering to understand ligand similarity and molecular docking study was done to understand binding orientations with catalytic site. Mechanism of enzyme action We have used two different approaches to decipher the reaction mechanism of the enzyme DHK. Fig 3. Reaction Mechanism of DHK and the effect of temperature on protein stability. Kinetic properties of the purified enzyme The kinetic properties of the purified enzyme were characterized by using standard substrate Ethyl 4-chloro acetoacetate and co-factor NADPH.
Substrate diversity The substrate diversity of the purified enzyme was determined by using spectrophotometric, both absorbance and fluorescence assays using Ethyl 4-chloro acetoacetate as a standard substrate.
Fig 9. Conclusion Our studies ascertain that the short-chain dehydrogenase reductase from Debaryomyceshansenii , DHK is an NADB-Rossmann oxidoreductase, and like other members of oxidoreductase family, can catalyze unfamiliar substrates. Supporting Information. S1 Fig. Oligomeric State determination of DHK. S2 Fig. Physico-chemical characterization of DHK.
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