Tài liệu Báo cáo khoa học: Functional expression and mutational analysis of flavonol synthase from

Functional expression and mutational analysis of flavonol synthase

from Citrus unshiu

Frank Wellmann1,*, Richard Lukacˇin1,*, Takaya Moriguchi2

, Lothar Britsch3

, Emile Schiltz4

and Ulrich Matern1

1

Institut fu¨r Pharmazeutische Biologie, Philipps-Universita¨t Marburg, Germany; 2

National Institute of Fruit Tree Science,

Ibaraki, Japan; 3

Merck kgaA, Scientific Laboratory Products, Darmstadt, Germany; 4

Institut fu¨r Organische Chemie

und Biochemie, Universita¨t Freiburg, Germany

Flavonols are produced by the desaturation of flavanols

catalyzed by flavonol synthase. The enzyme belongs to the

class of intermolecular dioxygenases which depend on

molecular oxygen and FeII/2-oxoglutarate for activity, and

have been in focus of structural studies recently. Flavonol

synthase cDNAs were cloned from six plant species, but

none of the enzymes had been studied in detail. Therefore, a

cDNA from Citrus unshiu (Satsuma mandarin) designated

as flavonol synthase was expressed in Escherichia coli, and

the purified recombinant enzyme was subjected to kinetic

and mutational chacterizations. The integrity of the recom￾binant synthase was revealed by a molecular ion from

MALDI-TOF mass spectrometry at m/z 37888 ± 40 (as

compared to 37899 Da calculated for the translated poly￾peptide), and by partial N-terminal sequencing. Maximal

flavonol synthase activity was observed in the range of

pH 5–6 with dihydroquercetin as substrate and a tempera￾ture optimum at about 37
C. Km values of 272, 11 and

36 lM were determined for dihydroquercetin, FeII and

2-oxoglutarate, respectively, with a sixfold higher affinity

to dihydrokaempferol (Km 45 lM). Flavonol synthase

polypeptides share an overall sequence similarity of 85%

(47% identity), whereas only 30–60% similarity were

apparent with other dioxygenases. Like the other dioxy￾genases of this class, Citrus flavonol synthase cDNA encodes

eight strictly conserved amino-acid residues which include

two histidines (His221, His277) and one acidic amino acid

(Asp223) residue for FeII-coordination, an arginine (Arg287)

proposed to bind 2-oxoglutarate, and four amino acids

(Gly68, His75, Gly261, Pro207) with no obvious function￾ality. Replacements of Gly68 and Gly261 by alanine reduced

the catalytic activity by 95%, while the exchange of these Gly

residues for proline completely abolished the enzyme activ￾ity. Alternatively, the substitution of Pro207 by glycine

hardly affected the activity. The data suggest that Gly68 and

Gly261, at least, are required for proper folding of the

flavonol synthase polypeptide.

Keywords: Citrus unshiu (Rutaceae); flavonoid biosyn￾thesis; flavonol synthase; functional expression; site-directed

mutagenesis.

Flavonoids fulfill vital functions in many plants beyond the

scope of pigmentation and ultraviolet screening, e.g. in

reproduction [1], in the defense against microbial pathogens

and insects or in auxin transport [2], and are accumulated

ubiquitously in flower and green tissues [1]. Their biosyn￾thesis proceeds from 4-coumaroyl- and malonyl-CoAs to

form naringenin chalcone [3] which is cyclized stereospeci￾fically to the flavanone (2S)-naringenin [3]. Naringenin may

be oxidized by flavone synthase (FNS) to yield the flavone

apigenin [4–6] or hydroxylated by flavanone 3b-hydroxylase

(FHT) to form a flavanol (syn. dihydroflavonol) [7–10], i.e.

dihydrokaempferol, which might be reduced subsequently

to a leucoanthocyanidin along the branch leading to

catechins and anthocyanidins [3] (Fig. 1). Alternatively,

flavonol synthase (FLS) catalyzes the oxidation of the

flavanol to a flavonol (Fig. 1). FLS had been reported

initially from irradiated parsley cells as a soluble dioxygen￾ase requiring 2-oxoglutarate and FeII/ascorbate for full

activity [11]. The activity was subsequently detected in

flower tissues of Matthiola incana [12], Petunia hybrida [13]

or Dianthus caryophyllus [14]. The first FLS cDNA was

cloned in 1993 from Petunia hybrida [15] and identified by

functional expression in yeast, while the FLS-antisense

transformation of petunia or tobacco intensified the red

flower pigmentation [15]. Further FLS cDNAs were

isolated later from Arabidopsis thaliana [16], Eustoma

grandiflorum, Solanum tuberosum [17], Malus domestica

and Matthiola incana, and approximately 85% similarity

was determined for the translated polypeptides, mostly in

the C-terminal 40% region based on total length of 335

residues. None of these enzymes has been satisfactorily

expressed and characterized.

Correspondence to U. Matern, Institut fu¨r Pharmazeutische Biologie,

Philipps-Universita¨t Marburg, Deutschhausstrasse 17A,

35037 Marburg, Germany.

Fax: + 49 6421 282 6678, Tel.: + 49 6421 282 2461,

E-mail: [email protected]

Abbreviations: ACC, aminocyclopropane-1-carboxylic acid;

DAOCS, deacetoxcephalosporin C synthase; FHT, flavanone

3b-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase;

IPNS, isopenicillin N synthase.

*Note: these authors contributed equally to the work presented.

Note: flavonol synthase NCBIdatabase accession numbers: Citrus

unshiu, AB011796; Eustoma grandiflorum, AAF64168; Malus domes￾tica, AAD26261; Matthiola incana, O04395.

(Received 17 April 2002, revised 4 July 2002, accepted 11 July 2002)

Eur. J. Biochem. 269, 4134–4142 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03108.x