no activity with substrate analogue 6,7-dihydropentalenolactone, because the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of pentalenolactone F. Enzyme-ligand interaction via three residues, F232, M77, and M81 that are unique to PntM and its orthologues and absent from essentially all other P450s
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purified wild-type enzyme enzyme PntM substrate-free, and PntM with bound substrate pentalenolactone F, product pentalenolactone, and substrate analogue 6,7-dihydropentalenolactone, and recombinant PntM F232L, M77S, M81A, M81C, and M81C-BME mutants with bound pentalenolactone F, sitting drop vapor diffusion method, mixing of 0.001 ml of 16.8 mg/mL protein in 10 mM Tris-HCl, 15 mM NaCl, and 10% glycerol, with 0.001 ml of reservoir solution containing 1.2 M sodium citrate, 10% glycerol, and 100 mM bicine, pH 9.0, 15°C, followed by cross-microseeding technique, X-ray diffraction structure determination and analysis at 1.54 A and 2.06-2.12 A resolution, respectively, molecular replacement using the crystal structure of polyene macrolide epoxidase PimD, PDB ID 2X9P, as search model
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 by nickel affinity chromatography, tag cleavage by thrombin, another step of nickel affinity chromatography, and gel filtration of the eluate, followed by ultrafiltration