Significance of C-terminal sequence elements for Petunia flavanone 3beta-hydroxylase activity.
Flavanone 3beta-hydroxylase (FHT), a 2-oxoglutarate-dependent dioxygenase (2-ODD), catalyzes the hydroxylation of (2S)-flavanones to (2R/3R)-dihydroflavonols in plants as a key step towards the biosynthesis of flavonols, anthocyanins and catechins. Crystallographic studies of 2-ODDs typically revealed a jelly roll in the enzyme core, and the C-terminus of the enzyme polypeptides was proposed to form a lid covering the active site cavity, thereby reducing the chances for oxidative or proteolytic damage and unfolding. Moreover, it has been proposed that in some cases the C-terminus is involved in substrate selectivity of 2-ODDs. In a systematic approach with highly active Petunia FHT, four C-terminally truncated enzyme forms were generated by deletion of five, 11, 24 or 29 amino acids. The recombinant FHTs preserved their substrate selectivity, but the specific activity decreased gradually with the extent of truncation. Then, an enzyme chimera was constructed by domain swapping replacing the C-terminal 52 amino acids of Petunia FHT by the equivalent region of flavonol synthase (FLS) from Citrus unshiu, an enzyme showing ambiguous FLS and FHT activity. The chimeric dioxygenase still revealed exclusively FHT activity, albeit at a moderate level only. The data predict that the selectivity of FHT is not governed by the C-terminal sequence accounting for about 13% of the enzyme polypeptide.
Wellmann
Frank
F
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17 A, 35037 Marburg, Germany.
Matern
Ulrich
U
Lukacin
Richard
R
D000595:Amino Acid Sequence; D002957:Citrus / Q000201:enzymology; D017124:Conserved Sequence; D044950:Flavanones / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D032306:Petunia / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion
D044950:Flavanones; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; C048797:flavanone 3-dioxygenase; C005273:naringenin