Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues.
A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.
Britsch
L
L
Biologisches Institut II der Universität, Lehrstuhl für Biochemie der Pflanzen, Freiburg, Germany.
Dedio
J
J
Saedler
H
H
Forkmann
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D007526:Isoelectric Point; D007930:Leucine / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019159:Nonheme Iron Proteins; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; D006639:Histidine; D006899:Mixed Function Oxygenases; C048797:flavanone 3-dioxygenase; D007930:Leucine; D004047:Diethyl Pyrocarbonate