Purification of recombinant flavanone 3 beta-hydroxylase from Petunia hybrida and assignment of the primary site of proteolytic degradation

Flavanone 3 beta-hydroxylase catalyzes the Fe-II/oxoglutarate-dependent hyd roxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybr ida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and c ommercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yi elds, two revised schemes of purification were developed for the recombinan t polypeptides, Applying a four-step protocol based on extraction and ion-e xchange chromatography at pH 7.5, the primary, catalytically inactive prote olytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on We stern blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the n ative 3 beta-hydroxylase. Thus, the native enzyme had been degraded by prot eolysis of a small carboxy-terminal portion, and the primary site of cleava ge must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with p hosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chro matography. This rapid, two-step purification resulted in a homogeneous 3 b eta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major break-through in mechanistic investiga tions of this class of labile dioxygenases, (C) 2000 Academic Press.