Site-directed mutagenesis of the active site serine290 in flavanone 3 beta-hydroxylase from Petunia hybrida

Flavanone 3 beta-hydroxylase (FHT) catalyzes a pivotal reaction in the form ation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In th e presence of oxygen and ferrous ions the enzyme couples the oxidative deca rboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase h ad been cloned from Petunia hybrida and expressed in Escherichia coil, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hyd roxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved ami no acids, including a strictly conserved serine residue (Ser290). This seri ne was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-depen dent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were s till significant, albeit greatly reduced to 20 and 8%, respectively, in com parison to the wild-type enzyme, whereas the activity of [Val290]FHT was ne gligible (about 1%). Kinetic analyses of purified wild-type and mutant enzy mes revealed the functional significance of Ser290 for 2-oxoglutarate-bindi ng. The spatial configurations of the related Fe(II)dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and p rovide the lead structures for the conformation of other dioxygenases. Circ ular dichroism spectroscopy was employed to compare the conformation of pur e flavanone 3 beta-hydroxylase with that of isopenicillin N synthase. A dou ble minimum in the far ultraviolet region at 222 nm and 208-210 nm and a ma ximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unc hanged after addition of either ferrous ions, 2-oxoglutarate or both of the se cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding.