Báo cáo khoa học: Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica doc

Although the methylenedioxy bridge-forming P450 CYP719 involved in berberine bio-synthesis has been cloned from Coptis japonica [Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, In

bridge-forming enzymes involved in stylopine biosynthesis

in Eschscholzia californica

Nobuhiro Ikezawa1, Kinuko Iwasa2and Fumihiko Sato1

1 Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Japan

2 Kobe Pharmaceutical University, Japan

Higher plants produce structurally divergent

chemi-cals, such as terpenoids, phenylpropanoids, and

alka-loids [1] Throughout human history, several plant

materials have been used in natural medicines

because of the pharmacological activities of these chemicals

Isoquinoline alkaloids are a large group of alkaloids, and include many pharmacologically useful compounds,

Keywords

alkaloid biosynthesis; cytochrome P450;

Eschscholzia californica; methylenedioxy

bridge-forming enzyme; stylopine synthase

Correspondence

F Sato, Division of Integrated Life Science,

Graduate School of Biostudies, Kyoto

University, Kyoto 606-8502, Japan

Fax: +81 75 753 6398

Tel +81 75 753 6381

E-mail: fsato@lif.kyoto-u.ac.jp

Note

The nucleotide sequences reported in this

paper have been submitted to the

DDBJ ⁄ GenBank ⁄ EMBL Data Bank under

the accession numbers AB126257

(CYP719A2) and AB126256 (CYP719A3)

(Received 6 September 2006, revised 23

November 2006, accepted 15 December

2006)

doi:10.1111/j.1742-4658.2007.05652.x

(S)-Stylopine is an important intermediate in the biosynthesis of benzophe-nanthridine alkaloids, such as sanguinarine Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine bio-synthesis has been cloned from Coptis japonica [Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem

278, 38557–38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis Two cyto-chrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C japonica CYP719 from cultured Esch-scholzia californicacells Heterologous expression in Saccharomyces

cerevisi-ae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline

to stylopine, but not cheilanthifoline synthase activity to convert scoulerine

to cheilanthifoline Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilan-thifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine An expression analysis in E californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and

S-adenosyl-l-methionine : 3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable Treatment

of E californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed

Abbreviations

BBE, berberine bridge enzyme; CHS, (S)-cheilanthifoline synthase; MeJA, methyl jasmonate; 4¢-OMT, S-adenosyl- L -methionine : 3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase; P450, cytochrome P450; RNAi, RNA interference; (S)-THB, (S)-tetrahydroberberine; (S)-THC, (S)-tetrahydrocolumbamine; STS, (S)-stylopine synthase.

such as morphine Despite their structural diversity,

most isoquinoline alkaloids are found in limited and

evolutionarily old taxonomic groups within the plant

kingdom, i.e Papaveraceae, Ranunculaceae,

Berberida-ceae, MenispermaBerberida-ceae, and a few other families Among

the wide array of chemicals produced in these plant

families, some are known to share, at least in part,

common biosynthetic pathways, e.g a key intermediate

(S)-reticuline [1]

The Papaveraceae plant California poppy,

Esch-scholzia californica, is a traditional medicinal plant of

Native Americans, and has been well investigated

because of the variety and pharmacological effects of

its alkaloids One of the alkaloids produced by this

plant is the antimicrobial sanguinarine, which had

been used as the component of toothpastes and

mouthwashes The biosynthetic pathways of

sanguina-rine, as well as other benzophenanthridine-type

alka-loids chelirubine and macarpine, have been completely

elucidated at the enzyme level [2,3] These highly

oxid-ized chemicals are biosynthesoxid-ized from two molecules

of l-tyrosine via the central intermediate (S)-stylopine

(S)-Stylopine, which has two methylenedioxy groups in

rings A and D of its protoberberine skeleton, is

pro-duced by a three-step conversion from the key

interme-diate (S)-reticuline; berberine bridge enzyme (BBE)

catalyzes oxidative cyclization of the N-methyl moiety

of (S)-reticuline to produce (S)-scoulerine [4,5], and

two cytochrome P450s (S)-cheilanthifoline synthase

(CHS; EC 1.14.21.2) and (S)-stylopine synthase (STS;

EC 1.14.21.1), sequentially form two methylenedioxy

bridges from (S)-scoulerine via (S)-cheilanthifoline to

(S)-stylopine [6,7] (Fig 1) These two P450s (CHS and

STS) have been studied in part by using microsomal

fractions of cultured E californica cells [7] In that

report, CHS and STS were found to be two

independ-ent P450s, although they both catalyze methylenedioxy

bridge-forming reactions using similar substrates

[(S)-scoulerine and (S)-cheilanthifoline] Because many

P450s, especially those involved in the detoxification of

xenobiotics, are known to have relatively broad

sub-strate specificity [8], CHS and STS reactions might be

catalyzed by a single P450 in different ways, or CHS

and STS may be two homologous P450s To clarify

this point, we tried to isolate methylenedioxy

bridge-forming P450 cDNA(s) from E californica

Previously, we isolated the first methylenedioxy

bridge-forming P450 (CYP719) cDNA, the protein of

which catalyzed the conversion of

(S)-tetrahydroco-lumbamine to (S)-tetrahydroberberine [(S)-canadine] in

berberine biosynthesis, from cultured cells of Japanese

goldthread Coptis japonica (Ranunculaceae) (Fig 1)

[9] C japonica produces a large amount of berberine,

which has been used as a medicine, for example as a stomach tonic, and the biosynthesis of berberine has been intensively studied [10,11] Because C japonica cells also produce coptisine [12], oxidized stylopine, it has been proposed that stylopine is biosynthesized in

C japonica However, C japonica CYP719 did not show CHS activity, although it is possible that it had STS activity, which we were not able to check because

of a lack of the substrate cheilanthifoline Because no previous report has described stylopine biosynthesis in

C japonica, it is possible that a coptisine biosynthetic pathway in C japonica may not be similar to the known stylopine biosynthetic pathway in E californica Because both CHS and STS catalyze methylenedioxy bridge-forming reactions using substrates quite similar

to that of C japonica CYP719 (Fig 1), we speculated that the primary structures of CHS and STS in E cali-fornica may be homologous to that of C japonica CYP719 Based on this idea, we amplified cDNA frag-ments that were homologous to C japonica CYP719 from single-stranded cDNAs prepared from cultured

E californica cells Next, we isolated two full-length cDNAs and characterized the enzymological activity of their recombinant proteins produced in a S cerevisiae expression system Both of these CYP719 homologs (CYP719A2 and CYP719A3) showed methylenedioxy bridge-forming activity to convert cheilanthifoline to stylopine (STS activity), but no activity to convert scoulerine to cheilanthifoline (CHS activity) In addi-tion, they used scoulerine as the substrate to produce nandinine, which has a methylenedioxy bridge in ring A (2,3-position) of scoulerine Based on their enzymological properties (substrate specificity and kin-etic parameters) and expression profiles, we discuss the physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis in E californica

Results

Isolation of cytochrome P450 cDNAs

To examine the presence of C japonica CYP719 homologs and their functions in stylopine biosynthesis

in E californica, cytochrome P450 (P450) cDNA frag-ments were amplified from single-stranded cDNAs, prepared from cultured E californica cells, using the degenerate primers designed for the P450-conserved regions specific to C japonica CYP719 [9] After nested PCR, clear PCR products at  280 bp were obtained and subcloned into pT7Blue T-vector Sequence analy-sis of two clones indicated that amplified cDNA frag-ments had an identical sequence that was highly homologous (73.2% identity for 280 bp PCR product)

Fig 1 Biosynthetic pathway for a variety of isoquinoline alkaloids (S)-Scoulerine is an intermediate at the branch point leading to benzophe-nanthridine alkaloids in E californica or berberine in C japonica The pathway from (S)-scoulerine to berberine is thought not to exist in

E californica, but is the main pathway in C japonica (surrounded by dotted line) CYP80B1, (S)-N-methylcoclaurine 3¢-hydroxylase; 4¢-OMT, S-adenosyl- L -methionine:3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase; BBE, berberine bridge enzyme.

to the corresponding region of C japonica CYP719

cDNA (designated EcCYPA) To isolate a full-length

clone of EcCYPA, 5¢- and 3¢-RACE were conducted

Although a full-length sequence of EcCYPA was

obtained, another sequence obtained with 5¢-RACE

showed a minor but distinct change from EcCYPA

(named EcCYPB) A full-length clone of EcCYPB was

also isolated with 5¢- and 3¢-RACE

Nucleotide sequences and predicted amino acid

sequences

Sequence analysis confirmed that the longest

full-length EcCYPA carried 1916 nucleotides, with an

ORF that encoded 495 amino acids (accession number

AB126256), and full-length EcCYPB contained 1718

nucleotides, with an ORF that encoded 495 amino

acids (accession number AB126257) EcCYPA and

EcCYPB were also highly homologous to C japonica

CYP719 at the amino acid level (64.8 and 65.1%

iden-tity, respectively), and the identity between EcCYPA

and EcCYPB was 84.6% These predicted amino acid sequences were classified into the same family as

C japonica CYP719 by the P450 nomenclature com-mittee (D.R Nelson, University of Tennessee, Mem-phis, TN) According to the suggestion of the nomenclature committee, C japonica CYP719 was renamed CYP719A1, and EcCYPA and EcCYPB were designated CYP719A3 and CYP719A2, respectively Sequence analysis also showed that CYP719A2 and CYP719A3 had conserved eukaryotic P450 regions: a helix K region, an aromatic region, and a heme-bind-ing region at the C-terminal end (Fig 2) In addition, their N-terminal regions contained hydrophobic domains that corresponded to the membrane anchor sequences of microsomal P450 species, which suggested that CYP719A2 and CYP719A3 were located in the endoplasmic reticulum Notably, a conserved threonine (corresponding to Thr252 of P450cam), which plays a significant role in oxygen activation [13], has been replaced by serine in both CYP719A2 and CYP719A3

as in C japonica CYP719A1 (Ser299 of both

Fig 2 Amino acid sequence alignment of the CYP719 family Boxes indicate conserved regions of eukaryotic P450, which are the helix K region, aromatic region, and heme-binding region The box with dotted line in the helix-K region indicates the conserved EXXR motif, which

is canonical to all P450s The arrows below the sequences indicate degenerate primers (Fw1, Fw2, and Rv1) used for the amplification of CYP719 homologous cDNA fragments from E californica The asterisk indicates the position of Ser299 of CYP719A2 and CYP719A3, which replaces the conserved threonine CYP719A1, methylenedioxy bridge-forming enzyme from C japonica (accession number AB026122).

CYP719A2 and CYP719A3) This substitution is only

found in a few species, such as Zea mays CYP88A1

(accession number U32579) and Nicotiana tabacum

CYP92A2 (accession number X95342)

Because the primary structures of CYP719A2 and

CYP719A3 were so similar to that of C japonica

CYP719A1, these P450s were speculated to catalyze

methylenedioxy bridge-forming reactions similar

to C japonica CYP719A1 Although C japonica

CYP719A1 is involved in berberine biosynthesis, it has

been reported that E californica does not produce a

detectable level of berberine [14–17] Thus, CYP719A2

and CYP719A3 appear to be involved in a biosynthetic

pathway other than berberine biosynthesis In contrast to

C japonica, E californica produces a large amount of the

benzophenanthridine alkaloid sanguinarine [14,15,18],

the biosynthesis of which requires two consecutive

methy-lenedioxy bridge-forming reactions from (S)-scoulerine

via (S)-cheilanthifoline to (S)-stylopine, i.e CHS and

STS reactions Because (S)-scoulerine and

(S)-cheilanthi-foline are structurally similar to

(S)-tetrahydrocolumb-amine [(S)-THC], the substrate of C japonica CYP719A1

[9], we examined whether CYP719A2 and CYP719A3

could function as CHS or STS

Heterologous expression of CYP719A2 and

CYP719A3 in S cerevisiae and their

enzymological activities

CYP719A2 and CYP719A3 were heterologously

expressed in S cerevisiae to characterize their

enzymo-logical activities S cerevisiae expression plasmids for

CYP719A2 and CYP719A3 were constructed and

introduced into the S cerevisiae strain AH22 Because

both CYP719A2 and CYP719A3 had putative

endo-plasmic reticulum-localizing signals, microsomal

frac-tions were prepared from recombinant S cerevisiae

cells, and their enzymatic activities were determined

using LC-MS

Microsomal fractions prepared from transgenic

S cerevisiae cells of both CYP719A2 and CYP719A3

showed STS activity, i.e they catalyzed

methylenedi-oxy bridge-formation from (R,S)-cheilanthifoline to

stylopine (Fig 3) The formation of stylopine from

cheilanthifoline was confirmed by direct comparison of

the reaction product with standard stylopine Although

the concentration of (R,S)-cheilanthifoline used for

enzyme assays was low (0.4 lm) due to its low

availab-ility, recombinant CYP719A2 and CYP719A3 were

able to convert it to stylopine, suggesting that both

CYP719A2 and CYP719A3 function as STS

However, when (S)-scoulerine was used as the

sub-strate, neither CYP719A2 nor CYP719A3 produced

cheilanthifoline, but rather produced another product (see below) By contrast, C japonica CYP719A1 does not utilize (S)-scoulerine [9], and did not utilize (R,S)-cheilanthifoline at a concentration of 0.4 lm (data not shown), despite its Km value of 0.27 lm toward (S)-THC [9] These results suggested that members of the CYP719 family (CYP719A1–A3) have different sub-strate specificities and⁄ or reaction activities

P450 natures of CYP719A2 and CYP719A3 The P450 natures of CYP719A2 and CYP719A3 were examined using reduced CO-difference spectra (Fig 4) Whereas CYP719A2 expressed in S cerevisiae cells showed a characteristic peak at 450 nm with a content

of 30 pmol P450 per mg microsomal protein, the micro-somal fraction of CYP719A3 expressed in S cerevisiae cells showed indistinguishable spectral pattern from that of control S cerevisiae cells harboring empty plasmid Because the expression level of CYP719A3 in

S cerevisiae cells was low, we characterized their P450 natures, particularly that of CYP719A3, based on their reaction dependencies on NADPH and oxygen using (S)-scoulerine as the substrate (Table 1) The absence of NADPH or removal of O2 by the glucose⁄ glucose oxidase⁄ catalase system [7] clearly inhibited the CYP719A2 and CYP719A3 activities

Substrate specificity and affinity of CYP719A2 and CYP719A3

To investigate which of these P450s, CYP719A2 or CYP719A3, functions mainly as STS, their detailed substrate specificity was examined using several types

of alkaloids harboring an ortho-methoxyphenol moiety

at 10 lm (Fig 5) LC-MS analysis showed that CYP719A2 and CYP719A3 could convert (S)-scouler-ine to a new product with a reduction of 2 m⁄ z This new product was suggested to be nandinine based

on its total m⁄ z and fragment ion patterns (data not shown) Nandinine is a scoulerine derivative with the methylenedioxy bridge in ring A (2,3-position), whereas cheilanthifoline is a derivative with the methylenedioxy bridge in ring D (9,10-position) Because neither CYP719A2 nor CYP719A3 catalyzed the methylenedioxy bridge-formation in ring D of scoulerine, they should strictly recognize ring A of the substrate Whereas CYP719A3 also converted (S)-THC to (S)-tetrahydroberberine [(S)-THB], CYP719A2 did not (data not shown) Neither CYP719A2 nor CYP719A3 reacted with any of seven other compounds [columbamine, reticuline, (R,S)-norreticuline, (S)-N-methylcoclaurine, (S)-coclaurine

(R,S)-6-O-methylnorlaudanosoline, and magnoflorine]

to make their corresponding products with a

methylen-edioxy bridge Also, neither CYP719A2 nor CYP719A3

catalyzed methylenedioxy bridge-formation with a (S)-scoulerine derivative, which was produced from (S)-scoulerine by S-adenosyl-l-methionine : coclaurine

TIC

m/z=326 m/z=324

min

(R,S)-cheilanthifoline;

m/z=326

H3CO

O O

0

10

8

6

4

2

0

height

( 106)

A

TIC

m/z=326 m/z=324

min

reaction product

0

10

8

6

4

2

0

height

( 106)

B

TIC

m/z=326 m/z=324

min

reaction product

0

10

8

6

4

2

0

height

( 106)

C

TIC

m/z=326 m/z=324

min

(R,S)-stylopine; m/z=324

O

O O

0

10

8

6

4

2

0

height

( 105)

D

Fig 3 LC-MS analysis of reaction products

of CYP719A2 and CYP719A3 using (R,S)-ch-eilanthifoline as the substrate Vector control reaction (A), CYP719A2 reaction (B), CYP719A3 reaction (C), and authentic (R,S)-stylopine (D) are shown TIC, total ion chromatogram.

N-methyltransferase [19] and was predicted to be

N-methylated (S)-scoulerine

Because the above results suggested that

(R,S)-chei-lanthifoline and (S)-scoulerine would be competitive

substrates for CYP719A2 and CYP719A3, we

exam-ined the reaction specificity using a mixture of these

compounds at 0.4 lm each As shown in Fig 6,

CYP719A2 exclusively used (R,S)-cheilanthifoline as

a substrate to produce stylopine By contrast,

CYP719A3 converted (R,S)-cheilanthifoline and

(S)-scoulerine to stylopine and nandinine at a

compar-able level (Fig 6) Furthermore, when

(R,S)-cheilan-thifoline and (S)-THC were used as mixed substrates

at 0.4 lm each, CYP719A3 showed comparable

methy-lenedioxy bridge-forming activities with both substrates

(Fig 6) These results indicated that CYP719A3 had

broader substrate specificity than CYP719A2, although

only the ring A (2,3-position) of the substrates was converted

Next, the substrate affinities of CYP719A2 and CYP719A3 were determined to examine their functions

in greater detail Enzyme activity was quantified by HPLC using (R,S)-scoulerine (for CYP719A2) or (S)-scoulerine (for CYP719A3) as the substrates, due

to the low availability of (R,S)-cheilanthifoline Both P450s showed Michaelis–Menten-type reaction kinetics when the substrate concentration was varied The kin-etic parameters, Kmand Vmax, of CYP719A2 were esti-mated to be 32 ± 7.1 lm and 0.43 ± 0.04 pmol productÆmin)1Æpmol)1P450, respectively, whereas those

of CYP719A3 were estimated to be 0.54 ± 0.03 lm and 4.5 ± 0.03 pmol productÆmin)1Æmg)1 microsomal protein These differences in the Kmvalues for scouler-ine explain the different reactivities of CYP719A2 and CYP719A3 for the mixture of cheilanthifoline and scoulerine (Fig 6), because both CYP719A2 and CYP719A3 catalyzed (R,S)-cheilanthifoline compar-ably at a substrate concentration of 0.4 lm (Fig 3)

Expression of CYP719A2 and CYP719A3 genes in

E californica plants: tissue specificity and their response to methyl jasmonate (MeJA)

We examined the accumulation of mRNA of CYP719A2 and CYP719A3 in E californica plant tissues (leaf, stem, and root) in comparison with three stylopine biosynthetic genes, CYP80B1 [(S)-N-methylcoclaurine 3¢-hydroxylase] [20], BBE [4,5], and S-adenosyl-l-methionine : 3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase (4¢-OMT) [21, 21a] (Fig 7) As a result, all of the genes examined showed similar expression patterns; the expression in root was greater than that in the leaf and stem, which suggests that both CYP719A2 and CYP719A3 are involved in alkaloid

-3) 4

-4 0

Wavelength (nm)

-3) 4

-4 0

Wavelength (nm)

Fig 4 Reduced CO-difference spectra of

CYP719A2 and CYP719A3 heterologously

expressed in S cerevisiae Reduced

CO-difference spectra were recorded using

microsomal fractions of recombinant

S cerevisiae (1 mg proteinÆmL)1) Solid line

is the spectrum of the microsomal fraction

from CYP719A2-expressing S cerevisiae (A)

or CYP719A3-expressing S cerevisiae (B).

Dotted line is the spectrum of the

micro-somal fraction from control S cerevisiae

transformed with empty plasmid.

Table 1 Methylenedioxy bridge-forming activity of CYP719A2 and

CYP719A3 toward (S)-scoulerine without NADPH or oxygen The

reaction was carried out under standard assay conditions except

for the amounts of enzyme preparations (200 n M P450 for

CYP719A2 or 1.1 lgÆlL)1 of microsomal protein for CYP719A3)

after removal of NADPH or oxygen with glucose ⁄ glucose oxidase.

U, units.

Addition

Relative activity CYP719A2

%

CYP719A3

%

40 m M glucose

+5 U glucose oxidase

+10 U catalase

40 m M glucose

+ boiled glucose oxidase

+10 U catalase

biosynthesis (Fig 7) Notably, CYP719A3 showed

extre-mely high expression in root compared with the other

tis-sues ( 30 times higher than in leaf), whereas other genes

only showed a moderate increase in expression in root ( 3–6 times higher than in leaf) When the probable relat-ive expression levels of CYP719A2 and CYP719A3 were

Fig 5 Chemical structures of compounds tested as potential substrates for CYP719A2 and CYP719A3 The apparent natural sub-strate (R,S)-cheilanthifoline is boxed Iso-quinoline alkaloids with an ortho-methoxyphenol moiety were tested as potential substrates The structure of

‘N-methylated (S)-scoulerine’ is speculated based on the reaction mechanism of S-adenosyl- L -methionine:coclaurine N-methyltransferase.

estimated from their copy numbers in the same samples

using standard curves drawn with plasmids

(pT7Blue-based constructs containing each gene) with quantitative

RT-PCR, the calculation indicated that the probable

relat-ive expression level of CYP719A3 against CYP719A2 was

 4 times higher in root, whereas it was < 2 times lower in

leaf and stem (data not shown) Because the expression of

CYP80B1, BBE, 4¢-OMT, CYP719A2, and CYP719A3

was all the highest in root, and sanguinarine is found

exclusively in root (data not shown), root is likely the main

organ for the biosynthesis and storage of sanguinarine in

E californica These results suggested that CYP719A3

might contribute more to stylopine biosynthesis than

CYP719A2 in root, whereas the physiological functions of

CYP719A2 and CYP719A3 need to be characterized

fur-ther

The effects of MeJA on the expression of CYP719A2

and CYP719A3 in E californica seedlings were

examined, because MeJA has been reported to induce the biosynthesis of various plant secondary metabolites [22–26], including benzophenanthridine alkaloid bio-synthesis in E californica [22] MeJA also induced the expression of the CYP80B1 and BBE genes [10], and the activities of CHS and STS [27] Our results indica-ted that treatment with MeJA increased the expression

of all five genes (CYP719A2, CYP719A3, CYP80B1, BBE, and 4¢-OMT), which suggests the cooperative regulation of these biosynthetic genes (Fig 8) How-ever, detailed characterization of their induction kinet-ics indicated minor but still marked differences; CYP719A2, CYP719A3, CYP80B1, and 4¢-OMT tran-scripts accumulated rapidly to reach the maximum level within 12 h, whereas the BBE transcript showed a slower and prolonged increase over 48 h (Fig 8) Concerning the differences between CYP719A2 and CYP719A3 transcripts, although their increases showed

A

B

C

D

E

F

Fig 6 CYP719A2 and CYP719A3 have different substrate selectivities toward (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumb-amine The reaction mixtures contained two substrates at 0.4 l M each For the (R,S)-cheilanthifoline and (S)-scoulerine assay, vector control reaction (A), CYP719A2 reaction (B), CYP719A3 reaction (C), and authentic (R,S)-stylopine (D) are shown For the (R,S)-cheilanthifoline and (S)-tetrahydrocolumbamine assay, CYP719A3 reaction (E) and authentic (R,S)-tetrahydroberberine (F) are shown Reaction products (1) and (3) showed the same retention times and m⁄ z-values as authentic (R,S)-stylopine and (R,S)-tetrahydroberberine, respectively, whereas the reaction product (2) was suggested to be nandinine by its total m ⁄ z and fragment ion patterns (data not shown).

similar kinetics, only CYP719A2 transcript showed a

rather constant expression level even at 48 h (Fig 8)

This result may reflect the physiologically different

functions of CYP719A2 and CYP719A3

Discussion

We isolated two full-length P450 cDNAs (CYP719A2

and CYP719A3) using degenerate primers, designed for

C japonica CYP719A1, from cultured E californica

cells (Fig 2) The primary structures of CYP719A2 and

CYP719A3 showed high similarity to C japonica

CYP719A1 (65.1 and 64.8% identity, respectively)

Their recombinant proteins produced in S cerevisiae

showed the activity of STS, which catalyzes the

methy-lenedioxy bridge-forming reaction from cheilanthifoline

to stylopine (Fig 3) By contrast, neither of them, nor

C japonica CYP719A1, showed the activity of CHS, which catalyzes the methylenedioxy bridge-forming reaction from scoulerine to cheilanthifoline (Fig 1) These results indicate that another enzyme is needed for CHS activity in stylopine biosynthesis, and supports

a previous report that CHS and STS are independent P450s [7]

Analysis of substrate specificity showed that CYP719A2 and CYP719A3 catalyzed methylenedioxy bridge-formation only in ring A of their substrates, e.g the conversion of scoulerine to nandinine (Fig 5) Our results indicate that members of the CYP719 family have strict substrate specificity for ring A (2,3-position)

C

Fig 7 Expression profiles of CYP719A2 and CYP719A3 genes in plant tissues of E cali-fornica Total RNA (1 lg) prepared from

E californica plant tissues (leaf, stem, and root) was used for DNase I treatment and reverse transcription Transcript levels of CYP80B1 (A), BBE (B), 4¢OMT (C), CYP719A2 (D), and CYP719A3 (E) were determined using quantitative RT-PCR with quadruple measurements The relative expression level shows the values standard-ized by that of leaf as 1 The error bar indi-cates the standard error of the mean.