Molecular characterization and functional expression of flavonol 6-hydroxylase

被引：26

作者：

Anzellotti D. ^{[1
]}

Ibrahim R.K. ^{[2
]}

机构：

[1] Molec. Membrane Dynamics Laboratory, Dept. of Anatomy and Cell Biology, McGill University, Montreal

[2] Ctr. for Struct. and Funct. Genomics, Department of Biology, Concordia University, Montreal

来源：

BMC Plant Biology | / 4卷 / 1期

关键词：

Flavonoid; Flavonol; Eukaryotic Expression System; Peptide Sequence Information; Internal Peptide Sequence;

D O I：

10.1186/1471-2229-4-20

中图分类号：

学科分类号：

摘要：

Background: Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis. Results: A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4′-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome. Conclusions: Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction. © 2004 Anzellotti and Ibrahim; licensee BioMed Central Ltd.

引用

共 35 条

[1]

Ibrahim R.K., Anzellotti D., Enzymatic Basis of Flavonoid Diversity, Integrative Phytochemistry: From Ethnobotany to Molecular Ecology, 37, pp. 1-36, (2003)

[2]

Ibrahim R.K., De Luca V., Khouri H., Latchinian L., Brisson L., Charest P.M., Enzymology and compartmentation of polymethylated flavonol glucosides in Chrysosplenium americanum, Phytochemistry, 26, pp. 1237-1249, (1987)

[3]

Anzellotti D., Ibrahim R.K., Novel flavonol 2-oxoglutarate dependent dioxygenase: Affinity purification, characterization, and kinetic properties, Arch Biochem Biophys, 382, pp. 161-172, (2000)

[4]

Prescott A.G., Lloyd M.D., The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism, Nat Prod Rep, 17, pp. 367-383, (2000)

[5]

Lukacin R., Urbanke C., Groning I., Matern U., The monomeric polypeptide comprises the functional flavanone 3b-hydroxylase from Petunia hybrida, FEBS Lett, 467, pp. 353-358, (2000)

[6]

Wellmann F., Lukacin R., Moriguchi T., Britsch L., Schiltz E., Matern U., Functional expression and mutational analysis of flavonol synthase from Citrus unshiu, Eur J Biochem, 269, pp. 4134-4142, (2002)

[7]

Saito K., Kobayashi M., Gong Z., Tanaka Y., Yamazaki M., Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase: Molecular cloning and functional expression of cDNA from a red forma of Perilla frutescens, Plant J, 17, pp. 181-189, (1999)

[8]

Gauthier A., Gulick P.J., Ibrahim R.K., cDNA cloning and characterization of a 3′/5′-O- methyltransferase for partially methylated flavonols from Chrysosplenium americanum, Plant Mol Biol, 32, pp. 1163-1169, (1996)

[9]

Lukacin R., Britsch L., Identification of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida flavanone 3b-hydroxylase, Eur J Biochem, 249, pp. 748-757, (1997)

[10]

Vazquez-Flota F., De Carolis E., Alarco A.M., De Luca V., Molecular cloning and characterization of desacetoxyvindoline-4- hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don, Plant Mol Biol, 34, pp. 935-948, (1997)

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