enantioselective 1,2-reduction of alpha,beta-unsaturated ketones and aryl ketones by perakine reductase. Perakine reductase catalyzes asymmetric reduction of enones and aromatic ketones, leading to alpha-allylic alcohols and alpha-aromatic alcohols. It is NADPH-dependent. Among the evaluated substances, 4'-nitroacetophenone is found to be the best ketone substrate, with yield and enantiomeric excess values exceeding 99%. Exclusive enantioselectivity of enzyme perakine reductase (PR)
perakine reductase catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis
the active site is formed by the catalytic tetrad Asp52, Tyr57, Lys84, and His126 at the center of the (alpha/beta)8-barrel structure. Upon NADPH binding, dramatic conformational changes and movements are observed: two additional beta-strands in the C terminus become ordered to form one alpha-helix, and a movement of up to 24 A occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity
molecular modeling of the reaction mechansim. In the model, the oxygen atom of the product's hydroxy group approaches the carboxamide group of NADPH, located within a hydrogen-bond distance of the gamma-N of the imidazole ring of His126. Substrate binding analysis: the large pocket is formed by the surface of residues Ile56, Ile87, Ile 90, His126, and Arg127, the three clustered Ile residues contributing to the hydrophobic property of the pocket may facilitate substrate binding, the small pocket comprises the surface of the Met21, Tyr57, and Lys84 residues, together with the nicotinamide riboside component of NADPH
three-dimensional structure modeling of wild-type and mutant apo methylated enzymes with (alpha/beta)8-barrels with eight parallel beta-strands and eight alpha-helices typical for AKR superfamily members, overview
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crystals of the purified and methylated enzyme are obtained by the hanging-drop vapour diffusion technique at 20Â°C with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 A, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 A
purified recombinant methylated His6-tagged enzyme wild-type and mutant A213W, hanging drop vapor diffusion method, mixing of 0.002 ml of 5.5 mg/ml protein in 10 mM Tris-HCl buffer, pH 7.0, 1 mM DTT, 10 mM EDTA, with 0002 ml reservoir solution containing 25% v/v PEG 4000, 0.1 mM sodium citrate, pH 5.6, equilibration against 1 ml of reservoir solution at 20Â°C for 7 days, X-ray diffraction structure determination and analysis at 2.31 A and 1.77 A resolution, respectively