Site‐directed mutagenesis of the active site serine290 in flavanone 3β‐hydroxylase from Petunia hybrida

摘要: Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In presence oxygen ferrous ions enzyme couples oxidative decarboxylation 2-oxoglutarate, releasing carbon dioxide succinate, with oxidation flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida expressed Escherichia coli, rapid isolation method for highly active, recombinant developed. Sequence alignments various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including strictly serine residue (Ser290). This was mutated threonine, alanine or valine, which represent acids found at corresponding sequence position other enzymes. mutant enzymes were E. coli purified homogeneity. catalytic activities [Thr290]FHT [Ala290]FHT still significant, albeit greatly reduced 20 8%, respectively, comparison wild-type enzyme, whereas activity [Val290]FHT negligible (about 1%). Kinetic analyses functional significance Ser290 2-oxoglutarate-binding. spatial configurations related Fe(II)-dependent isopenicillin N deacetoxycephalosporin C synthases have reported recently provide lead structures conformation dioxygenases. Circular dichroism spectroscopy employed compare pure flavanone that synthase. A double minimum far ultraviolet region 222 nm 208-210 maximum 191-193 are characteristic alpha-helical regions observed, spectra two fully matched revealing their close structural relationship. Furthermore, spectrum remained unchanged after addition either ions, 2-oxoglutarate both these cofactors, ruling out significant conformational change on cofactor-binding.