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
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 20°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 20°C for 7 days, X-ray diffraction structure determination and analysis at 2.31 A and 1.77 A resolution, respectively