Tài liệu Báo cáo khoa học: Octaketide-producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins pdf

Although HpPKS2 was found to have octaketide synthase activity, production of emodin anthrone, a supposed octaketide precursor of hypericins, was not detected.. The type III PKS involved

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Hypericum perforatum is expressed in dark glands

accumulating hypericins

Katja Karppinen1, Juho Hokkanen2,*, Sampo Mattila2, Peter Neubauer3 and Anja Hohtola1

1 Department of Biology, University of Oulu, Finland

2 Department of Chemistry, University of Oulu, Finland

3 Department of Process and Environmental Engineering, University of Oulu, Finland

Hypericum perforatum L., St John’s wort, is a

medici-nal plant that is widely utilized for the treatment of

mild to moderate depression [1,2] Hypericins, a

group of red-pigmented naphthodianthrones including

hypericin and pseudohypericin, as well as their

intimate precursors protohypericin and

proto-pseudohypericin (Fig 1), are considered the principal

agents in the range of biological activities reported for

H perforatum [3–5] Hypericins, together with other

bioactive compounds of the crude plant extract, such

as hyperforins and ﬂavonoids, have been found to con-tribute to the antidepressant activity of the plant [1–3] Hypericin is the most potent natural photosensitizer described to date, and its photodynamic activities allow hypericin to also act as an antiviral and antitu-moral agent [3,6–8] In H perforatum, hypericins have been suggested to act as the plant’s defence against insects [9]

H perforatum is characterized by the presence of dark glands in the aerial parts of the plant [10–13]

Keywords

dark glands; hypericin; octaketide synthase;

St John’s wort (Hypericum perforatum L.);

type III polyketide synthase

Correspondence

K Karppinen, Department of Biology,

University of Oulu, PO Box 3000, FIN-90014

Oulu, Finland

Fax: +358 8 553 1061

Tel: +358 8 553 1544

E-mail: katja.karppinen@oulu.fi

*Present address

Novamass Ltd, Oulu, Finland

Database

Nucleotide sequence data have been

submitted to the DDBJ⁄ EMBL ⁄ GenBank

databases under the accession number

EU635882

(Received 26 April 2008, revised 18 June

2008, accepted 27 June 2008)

doi:10.1111/j.1742-4658.2008.06576.x

Hypericins are biologically active constituents of Hypericum perforatum (St John’s wort) It is likely that emodin anthrone, an anthraquinone precursor of hypericins, is biosynthesized via the polyketide pathway by type III polyketide synthase (PKS) A PKS from H perforatum, HpPKS2, was investigated for its possible involvement in the biosynthesis of hyperic-ins Phylogenetic tree analysis revealed that HpPKS2 groups with function-ally divergent non-chalcone-producing plant-speciﬁc type III PKSs, but it

is not particularly closely related to any of the currently known type III PKSs A recombinant HpPKS2 expressed in Escherichia coli resulted in an enzyme of 43 kDa The puriﬁed enzyme catalysed the condensation of acetyl-CoA with two to seven malonyl-CoA to yield tri- to octaketide prod-ucts, including octaketides SEK4 and SEK4b, as well as heptaketide aloe-sone Although HpPKS2 was found to have octaketide synthase activity, production of emodin anthrone, a supposed octaketide precursor of hypericins, was not detected The enzyme also accepted isobutyryl-CoA, benzoyl-CoA and hexanoyl-CoA as starter substrates producing a variety

of tri- to heptaketide products In situ RNA hybridization localized the HpPKS2 transcripts in H perforatum leaf margins, ﬂower petals and stamens, speciﬁcally in multicellular dark glands accumulating hypericins Based on our results, HpPKS2 may have a role in the biosynthesis of hypericins in H perforatum but some additional factors are possibly required for the production of emodin anthrone in vivo

Abbreviations

CHS, chalcone synthase; DIG, digoxigenin; IPTG, isopropyl thio-b- D -galactoside; OKS, octaketide synthase; PCS, pentaketide chromone synthase; PKS, polyketide synthase; STS, stilbene synthase.

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Dark glands appear as black or dark red multicellular

nodules that occur near the leaf margins, stems, ﬂower

petals and stamens [10–12,14] Hypericum species with

dark glands are known to produce hypericins [15] The

correlation between the concentrations of hypericins

and the existence of dark glands in H perforatum

tis-sues has shown the presence of these red pigments in

the glands [12,16,17] Hypericin has also been shown

to accumulate in red glands in the sepals in H elodes

[15] It has been proposed that hypericins not only

accumulate, but are also biosynthesized in the dark

glands [12,18] The localization of hypericins in the

nodular structures is considered to have evolved as a

mechanism for the plant to avoid the potential

auto-toxicity of these compounds [19]

The biosynthesis of hypericins is currently poorly

understood, but the polyketide pathway is likely to

play a central role [12,20] Plant-speciﬁc type III

poly-ketide synthases (PKSs) are involved in the

biosynthe-sis of a large variety of plant secondary metabolites,

including chalcones, stilbenes, benzophenones,

acri-dones, phloroglucinols and benzalacetone derivatives

[21] The enzymes catalyse the formation of complex

natural products by condensing various CoA-thioesters

with malonyl-CoA in a reaction sequence that closely

parallels fatty acid biosynthesis [22] The functional

diversity of simple homodimeric type III PKSs is

derived from small differences in the active site that

inﬂuence the substrate speciﬁcities, the number of

con-densation reactions and the mechanisms of cyclization

reactions [22,23] In some cases, the reaction

inter-mediates are also modiﬁed by interaction with other

enzymes [23] The type III PKS involved in the

biosyn-thesis of hypericins has been suggested to condensate

one molecule of acetyl-CoA with seven molecules of

malonyl-CoA to form an octaketide chain that subse-quently undergoes cyclizations and decarboxylation, leading to the formation of emodin anthrone (Fig 1),

a precursor of hypericins [3,12,20] However, there are

no reports on the characterization of the type III PKS with octaketide synthase (OKS) activity which is responsible for the formation of emodin anthrone The ﬁnal stages of hypericin biosynthesis are conducted by the gene product of hyp-1, encoding for the phenolic coupling protein that catalyses the oxidative dimeriza-tion of emodin anthrone to hypericin [3,20]

To date, four different PKS family genes have been cloned from the genus Hypericum Chalcone synthase (CHS) and benzophenone synthase have been cloned from both H androsaemum [24] and H perforatum [25] In addition, in a recent study, we described the cloning of two previously uncharacterized cDNAs from H perforatum encoding for PKSs, designated as HpPKS1 and HpPKS2 [25] Expression of HpPKS2 was found to correlate with the concentration of hyp-ericins in H perforatum tissues and HpPKS2 is thus a candidate gene for the biosynthesis of hypericins [25]

In this study, the role of H perforatum HpPKS2 is investigated in more detail We describe the functional characterization of HpPKS2 and the exact localization

of HpPKS2 transcripts in H perforatum leaves and ﬂower buds using in situ RNA hybridization HpPKS2 was found to be an OKS and is speciﬁcally expressed in the dark glands accumulating hypericins Thus, the results imply that HpPKS2 may have a role in the bio-synthesis of hypericins in H perforatum The failure of HpPKS2 to catalyse the formation of emodin anthrone

in vitro, but produce other octaketides instead, is dis-cussed in terms of a possible need for some additional factors for the production of emodin anthrone

CoAS

O

Acetyl-CoA

+ 7 × malonyl-CoA

OKS

O

Emodin

O

Oxidation

O O O O

SEnz O

SEK4

O O O

O O OH

O

SEK4b

O O O O

O OH O

in vitro

Cyclizations, decarboxylation

in vivo

OH O OH

R

OH O OH O

R = CH3, Hypericin

R = CH2OH, Pseudohypericin

R = CH3, Protohypericin

R = CH2OH, Protopseudohypericin

OH O OH

CH3 R

OH O OH

O O

Oxidative dimerization

3 2

O

Emodin anthrone

Fig 1 Putative reaction of OKS involved in the biosynthesis of hypericins in H perforatum In vivo OKS is suggested to condensate one ace-tyl-CoA with seven malonyl-CoA to form an octaketide chain that subsequently undergoes cyclizations and decarboxylation to form emodin anthrone It is possible that in vitro OKS affords shunt products SEK4 and SEK4b in the absence of some additional, yet unidentified factors.

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During ampliﬁcation of the HpPKS2 coding sequence

from H perforatum, several cDNA clones of HpPKS2

that differed slightly from one another were

encoun-tered The deduced amino acid sequences of the clones

shared 99–100% identity The HpPKS2 clone that was

found to be the most abundant of the different cDNA

clones in H perforatum was selected for investigation

in this study The nucleotide sequence of the clone has

been deposited in GenBank under the accession

num-ber EU635882 However, because the HpPKS2 clones

showed such high sequence similarity and thus their

expression in H perforatum tissues could not be

distin-guished from each other, the general name HpPKS2 is

used in this study

Phylogenetic analysis

The overall similarity of the deduced amino acid

sequence of HpPKS2 with other type III PKS family

proteins was investigated using a neighbor-joining tree (Fig 2) Phylogenetic analysis showed that the members of the plant-speciﬁc type III PKSs grouped into CHSs and non-CHSs, except stilbene synthases (STSs) from Fabaceae and Gymnosperms In these cases, the STSs were closer to CHSs of the same or related species than other non-chalcone-forming PKSs HpPKS2 grouped with functionally divergent non-chalcone-forming plant-speciﬁc type III PKSs, including OKS and pentaketide chromone synthase (PCS) from Aloe arborescens [26,27] However, HpPKS2 was positioned on a sub-branch of its own without any particularly closely related proteins

Expression of HpPKS2 in Escherichia coli

To study the enzymatic function of HpPKS2 in more detail, the coding region of the HpPKS2 cDNA was functionally expressed in Escherichia coli strain M15 [pREP4] with pQE30 vector When E coli cells harbouring the recombinant plasmid were grown at

Oryza sativa CHS (AB000801) Zea mays CHS (X60205) Ruta graveolens CHS (AJ297789) Gerbera hybrida CHS (Z38096) Arabidopsis thaliana CHS (AF112086) Vitis vinifera CHS (X75969) Hypericum androsaemum CHS (AF315345) Sorbus aucuparia CHS (DQ286037) Camellia sinensis CHS (D26593) Petunia hybrida CHS (X04080) Hydrangea macrophylla CHS (AB011467) Picea mariana CHS (AF227627) Pinus sylvestris CHS (X60754) Pinus sylvestris STS (S50350) Pueraria lobata CHS (D10223) Phaseolus vulgaris CHS (X06411) Pisum sativum CHS (X63333) Medicago sativa CHS (L02902) Glycine max CHS (X53958) Arachis hypogaea STS (L00952) Vitis vinifera STS (S63221) Rheum palmatum BAS (AF326911) Humulus lupulus VPS (AB015430) Hydrangea macrophylla CTAS (AB011468) Hydrangea macrophylla STCS (AF456445) Ruta graveolens ACS (AJ297788) Gerbera hybrida 2-PS (Z38097) Rheum palmatum ALS (AY517486) Plumbago indica PKS (AB259100) Phalaenopsis sp BBS (X79903) Bromheadia finlaysoniana BBS (AJ131830) Sorbus aucuparia BIS (DQ286036) Hypericum androsaemum BPS (AF352395) Wachendorfia thyrsiflora PKS1 (AY727928) Ipomoea purpurea CHS-B (U15947) Ipomoea purpurea CHS-A (U15946) Aloe arborescens PCS (AY823626) Aloe arborescens OKS (AY567707) Hypericum perforatum HpPKS2 (EU635882) Aspergillus oryzae csyB (AB206759) Aspergillus oryzae csyA (AB206758) Fusarium graminearum FG08378.1 (XM_388554) Magnaporthe grisea MG04643.4 (XM_362198) Streptomyces griseus RppA (AB018074)

100

99

94

90 57 54

51

87

100

98 100

100

0,1

Fabaceae Gymnosperms

divergent PKSs

CHSs

plants

fungi bacteria

Oryza sativa CHS (AB000801) Zea mays CHS (X60205) Ruta graveolens CHS (AJ297789) Gerbera hybrida CHS (Z38096) Arabidopsis thaliana CHS (AF112086) Vitis vinifera CHS (X75969) Hypericum androsaemum CHS (AF315345) Sorbus aucuparia CHS (DQ286037) Camellia sinensis CHS (D26593) Petunia hybrida CHS (X04080) Hydrangea macrophylla CHS (AB011467) Picea mariana CHS (AF227627) Pinus sylvestris CHS (X60754) Pinus sylvestris STS (S50350) Pueraria lobata CHS (D10223) Phaseolus vulgaris CHS (X06411) Pisum sativum CHS (X63333) Medicago sativa CHS (L02902) Glycine max CHS (X53958) Arachis hypogaea STS (L00952) Vitis vinifera STS (S63221) Rheum palmatum BAS (AF326911) Humulus lupulus VPS (AB015430) Hydrangea macrophylla CTAS (AB011468) Hydrangea macrophylla STCS (AF456445) Ruta graveolens ACS (AJ297788) Gerbera hybrida 2-PS (Z38097) Rheum palmatum ALS (AY517486) Plumbago indica PKS (AB259100) Phalaenopsis sp BBS (X79903) Bromheadia finlaysoniana BBS (AJ131830) Sorbus aucuparia BIS (DQ286036) Hypericum androsaemum BPS (AF352395) Wachendorfia thyrsiflora PKS1 (AY727928) Ipomoea purpurea CHS-B (U15947) Ipomoea purpurea CHS-A (U15946) Aloe arborescens PCS (AY823626) Aloe arborescens OKS (AY567707) Hypericum perforatum HpPKS2 (EU635882) Aspergillus oryzae csyB (AB206759) Aspergillus oryzae csyA (AB206758) Fusarium graminearum FG08378.1 (XM_388554) Magnaporthe grisea MG04643.4 (XM_362198) Streptomyces griseus RppA (AB018074)

100

99

94

90 57 54

51

87

100

98 100

100

0,1

CHSs

plants

fungi bacteria

CHSs

Functionally

CHSs

plants

fungi bacteria

plants

fungi bacteria

Fig 2 Phylogenetic analysis of type III PKS

enzymes The tree was constructed using

the neighbor-joining algorithm The numbers

at the forks are bootstrap values that

indicate the per cent values for obtaining

this particular branching in 1000 replicates;

only values > 50% are shown The indicated

scale represents 0.1 amino acid

substitu-tions per site The GenBank accession

num-bers are followed by the names of the

species ACS, acridone synthase; ALS,

aloe-sone synthase; BAS, benzalacetone

syn-thase; BBS, bibenzyl synsyn-thase; BIS,

biphenyl synthase; BPS, benzophenone

syn-thase; CHS, chalcone synsyn-thase; CTAS,

4-coumaroyltriacetic acid synthase; OKS,

octaketide synthase; PCS, pentaketide

chromone synthase; 2-PS, 2-pyrone

synthase; STCS, stilbene carboxylate

synthase; STS, stilbene synthase; VPS,

valerophenone synthase.

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37C after induction with isopropyl

thio-b-d-galacto-side (IPTG), all the induced HpPKS2 proteins

became insoluble Similar phenomena have been

reported previously in the expression of some

plant-speciﬁc type III PKSs in E coli [28,29], and in many

cases, a low temperature has been used to obtain

recombinant PKS in a soluble form [28,30–32]

There-fore, the culture temperature was lowered to 16C

after induction with IPTG Under these culture

condi-tions, IPTG-induced E coli cells produced the soluble

HpPKS2 protein, as shown on SDS⁄ PAGE gel

(Fig 3) Because the recombinant HpPKS2 protein

contained an additional hexahistidine tag at the

N-terminus, it enabled us to obtain the enzyme with

high purity after puriﬁcation with Ni-NTA agarose

After puriﬁcation, commonly 2.5 mg of pure

recombinant HpPKS2 was obtained from 1 g of

E coli cell pellet The puriﬁed enzyme gave a band

with a molecular mass of 43 kDa on SDS ⁄ PAGE

gel (Fig 3)

Enzyme activity of recombinant HpPKS2

The enzymatic activity of the puriﬁed recombinant

HpPKS2 was tested for suggested emodin

anthrone-forming activity by using acetyl-CoA as a starter

sub-strate Some of the products (Fig 4), determined by

UPLC⁄ ESIMS, were simple a-pyrones with a linear

keto side chain (A1, A2, A3) showing loss of CO2

from the parent ion ([M-H-44])) in the negative

ioni-zation mode (Fig 5A) Other characteristic fragments

at m⁄ z 125 corresponding to [C6H5O3]) (pyrone moi-ety) and m⁄ z 167 corresponding to [C8H7O4]) were also detected for some a-pyrones, depending on the chain length of the particular compound Octaketides SEK4 (A4) and SEK4b (A7), as well as heptaketide aloesone (A9), were also found from incubations The proposed fragmentation patterns of SEK4 and SEK4b

in the negative ionization mode are presented in Fig 5B,C, respectively A heptaketide aloesone was identified based on its UV spectrum and the structure was confirmed by its fragmentation in the positive ionization mode (Fig 5D) HpPKS2 also produced pentaketide chromone A8 (2,7-dihydroxy-5-methyl-chromone) and heptaketide chromone A10 [1-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)pentane-2,4-dione] The structure A8 was identified based on its UV spectrum, exact mass and retention behaviour [26] Structure A10 was identified based on its exact mass and fragment ion at m⁄ z 189 (loss of acyl side chain)

in ESI) conditions Also, heptaketide phenylpyrone A6 [6-(2,4-dihydroxy-6-(2-oxopropyl)phenyl)-4-hydro-xy-2H-pyran-2-one] showing loss of CO2 from the parent ion ([M-H-44])), but no other fragments under ESI) conditions, was identiﬁed Although HpPKS2 showed the expected OKS activity, emodin anthrone,

a supposed octaketide precursor of hypericins, was not detected

Recombinant HpPKS2 was also examined for its ability to use other CoA-thioesters as starter sub-strates It was found that HpPKS2 accepted all tested starter units (isobutyryl-CoA, benzoyl-CoA and hexa-noyl-CoA) to produce a variety of tri- to heptaketide products (Fig 4), most of which were identiﬁed as a-pyrones with a linear keto side chain (B1, B2, B3, B4, C1, C2, C3, C5, C7, D1, D2, D4, D6) In addition, chromones B8 [1-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-5-methylhexane-2,4-dione] and C6 [5,7-dihydroxy-2-(2-oxo-2-phenylethyl)-4H-chromen-4-one], phloroglucinols B6 [6-methyl-1-(2,4,6-trihydroxyphe-nyl)heptane-1,3,5-trione] and D5 [1-(2,4,6-trihydroxy-phenyl)decane-1,3,5-trione], as well as phenylpyrones B5 [6-(2,4-dihydroxy-6-(3-methyl-2-oxobutyl)phenyl) -4-hydroxy-2H-pyran-2-one], C4 [6-(3,5-dihydroxy biphenyl-2-yl)-4-hydroxy-2H-pyran-2-one] and D3 [6-(2,4-dihydroxy-6-pentylphenyl)-4-hydroxy-2H-pyran-2-one] were detected Identiﬁcation of the compounds was made based on their similar fragmentation, UV characteristics and retention behaviour compared with the corresponding products obtained using acetyl-CoA as a starter substrate None of the above-men-tioned products was found from negative control reactions that contained heat-denatured enzyme with corresponding substrates

116.0 66.2 45.0 35.0

25.0

18.4 14.4

Fig 3 SDS ⁄ PAGE analysis of recombinant HpPKS2 expressed in

E coli (1) Total proteins from E coli without induction, (2) total

pro-teins from E coli induced with IPTG, (3) soluble propro-teins, (4)

puri-fied recombinant HpPKS2 protein, (M) protein molecular mass

marker, with sizes (kDa) indicated at the right.

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Hexaketides

Pentaketides

Substrate

Tetraketides

Triketides

O O

OH

C2

RTUPLC= 1.50 min m/z 355 [M-H]

-λmax274 nm

O O

OH

-λ max 307 nm

C4

O O

OH

O O C7

RT UPLC = 2.63 min m/z 271 [M-H]

-λmax 316 nm

O

O OH

O O C6

-λmax242, 285, 340 nm O

O

OH

C1

-λmax264 nm

CoAS O Benzoyl-CoA

O O

OH O C3

-λmax246, 284 nm

O O

OH

-λ max 219, 233, 318 nm

C5

O O

OH

D3

-λ max 298 nm

O O O

OH O D6

-λmax281 nm

O

OH

O

D1

-λmax261 nm

OH O O O D5

-λmax 288 nm

CoAS O Hexanoyl-CoA

O

OH

O O D2

-λmax284 nm

O O

OH D4

-λ max 227, 284 nm

O O

OH

O B5

-λmax238, 286 nm

OH O O O

-λ max 238, 287 nm

B6

O O

OH

B2

-λ max 264 nm

O O

OH

B1

-λmax262 nm

O O

OH O

O O B8

-λ max 230, 281, 324, 337, 404 nm

CoAS O Isobutyryl-CoA

O O

OH O B3

-λmax284 nm

O O

OH

-λ max 225, 284 nm

B4

Octaketides

O O O O O OH O A7

-λ max 230, 280 nm

O O O

O O OH

O A4

-λmax284 nm

O O

OH

O A6

-λmax282 nm

CoAS O Acetyl-CoA

O O

OH

-λ max 270 nm

A1

O O

OH O A2

-λmax 285 nm O

O

OH O A8

-λmax309 nm

-λ max 283 nm

OH

O

O O A9

-λ max 243, 251, 292 nm O

O O

OH

O O A10

-λmax 235, 276 nm

Heptaketides

Hexaketides

Pentaketides

Substrate

Tetraketides

Triketides

O O

OH

C2

-λmax274 nm

O O

OH

-λ max 307 nm

C4

O O

OH

O O C7

-λmax 316 nm

O

O OH

O O C6

-λmax242, 285, 340 nm O

O

OH

C1

-λmax264 nm

CoAS O Benzoyl-CoA

O O

OH O C3

-λmax246, 284 nm

O O

OH

-λ max 219, 233, 318 nm

C5

O O

OH

D3

-λ max 298 nm

O O O

OH O D6

-λmax281 nm

O

OH

O

D1

-λmax261 nm

OH O O O D5

-λmax 288 nm

CoAS O Hexanoyl-CoA

O

OH

O O D2

-λmax284 nm

O O

OH D4

-λ max 227, 284 nm

O O

OH

O B5

-λmax238, 286 nm

OH O O O

-λ max 238, 287 nm

B6

O O

OH

B2

-λ max 264 nm

O O

OH

B1

-λmax262 nm

O O

OH O

O O B8

-λ max 230, 281, 324, 337, 404 nm

O O

OH O B3

-λmax284 nm

O O

OH

-λ max 225, 284 nm

B4

Octaketides

O O O O O OH O A7

-λ max 230, 280 nm

O O O

O O OH

O A4

-λmax284 nm

O O

OH

O A6

-λmax282 nm

CoAS O Acetyl-CoA

O O

OH

-λ max 270 nm

A1

O O

OH O A2

-λmax 285 nm O

O

OH O A8

-λmax309 nm

-λ max 283 nm

OH

O

O O A9

-λ max 243, 251, 292 nm O

O O

OH

O O A10

-λmax 235, 276 nm

Heptaketides

Hexaketides

Pentaketides

Substrate

Tetraketides

Triketides

Heptaketides

Hexaketides

Pentaketides

Substrate

Tetraketides

Triketides

O O

OH

C2

-λmax274 nm

O O

OH

-λ max 307 nm

C4

O O

OH

O O C7

-λmax 316 nm

O

O OH

O O C6

-λmax242, 285, 340 nm O

O

OH

C1

-λmax264 nm

CoAS O Benzoyl-CoA

O O

OH O C3

-λmax246, 284 nm

O O

OH

-λ max 219, 233, 318 nm

C5

O O

OH

D3

-λ max 298 nm

O O O

OH O D6

-λmax281 nm

O

OH

O

D1

-λmax261 nm

OH O O O D5

-λmax 288 nm

CoAS O Hexanoyl-CoA

O

OH

O O D2

-λmax284 nm

O O

OH D4

-λ max 227, 284 nm

O O

OH

O B5

-λmax238, 286 nm

OH O O O

-λ max 238, 287 nm

B6

O O

OH

B2

-λ max 264 nm

O O

OH

B1

-λmax262 nm

O O

OH O

O O B8

-λ max 230, 281, 324, 337, 404 nm

O O

OH O B3

-λmax284 nm

O O

OH

-λ max 225, 284 nm

B4

Octaketides

O O O O O OH O A7

-λ max 230, 280 nm

O O O

O O OH

O A4

-λmax284 nm

O O

OH

O A6

-λmax282 nm

CoAS O Acetyl-CoA

O O

OH

-λ max 270 nm

A1

O O

OH O A2

-λmax 285 nm O

O

OH O A8

-λmax309 nm

-λ max 283 nm

OH

O

O O A9

-λ max 243, 251, 292 nm O

O O

OH

O O A10

-λmax 235, 276 nm

O O

OH

C2

-λmax274 nm

O O

OH

-λ max 307 nm

C4

O O

OH

O O C7

-λmax 316 nm

O

O OH

O O C6

-λmax242, 285, 340 nm O

O

OH

C1

-λmax264 nm

CoAS O Benzoyl-CoA

O O

OH O C3

-λmax246, 284 nm

O O

OH

-λ max 219, 233, 318 nm

C5

O O

OH

C2

-λmax274 nm

O O

OH

C2

RTUPLC= 1.50 min m/z 355 [M-H] –

λmax274 nm

O O

OH

-λ max 307 nm

C4 O O

OH

λ max 307 nm

C4

O O

OH

O O C7

-λmax 316 nm

O O

OH

O O C7

RT UPLC = 2.63 min m/z 271 [M-H] –

λmax 316 nm

O

O OH

O O C6

-λmax242, 285, 340 nm

O

O OH

O O C6

RT UPLC = 2.54 min m/z 295 [M-H] –

λmax242, 285, 340 nm O

O

OH

C1

-λmax264 nm

O O

OH

C1

RT UPLC = 1.25 min m/z 313 [M-H] –

λmax264 nm

CoAS O Benzoyl-CoA

O O

OH O C3

-λmax246, 284 nm

O O

OH O C3

λmax246, 284 nm

O O

OH

-λ max 219, 233, 318 nm

OH

λ max 219, 233, 318 nm

C5

O O

OH

D3

-λ max 298 nm

O O O

OH O D6

-λmax281 nm

O

OH

O

D1

-λmax261 nm

OH O O O D5

-λmax 288 nm

CoAS O Hexanoyl-CoA

O

OH

O O D2

-λmax284 nm

O O

OH D4

-λ max 227, 284 nm

O O

OH

D3

-λ max 298 nm

O O

OH

D3

λ max 298 nm

O O O

OH O D6

-λmax281 nm

O O O

OH O D6

RT UPLC = 3.16 min m/z 265 [M-H] –

λmax281 nm

O

OH

O

D1

-λmax261 nm

O

OH

O

D1

λmax261 nm

OH O O O D5

-λmax 288 nm

OH O O O D5

RT UPLC = 3.13 min m/z 307 [M-H] –

λmax 288 nm

CoAS O Hexanoyl-CoA

O

OH

O O D2

-λmax284 nm

O

OH

O O D2

λmax284 nm

O O

OH D4

-λ max 227, 284 nm

O O

OH D4

λ max 227, 284 nm

O O

OH

O B5

-λmax238, 286 nm

OH O O O

-λ max 238, 287 nm

B6

O O

OH

B2

-λ max 264 nm

O O

OH

B1

-λmax262 nm

O O

OH O

O O B8

-λ max 230, 281, 324, 337, 404 nm

O O

OH O B3

-λmax284 nm

O O

OH

-λ max 225, 284 nm

B4

O O

OH

O B5

-λmax238, 286 nm

O O

OH

O B5

λmax238, 286 nm

OH O O O

-λ max 238, 287 nm

B6

OH O O O

λ max 238, 287 nm

B6

O O

OH

B2

-λ max 264 nm

O O

OH

B2

λ max 264 nm

O O

OH

B1

-λmax262 nm

O O

OH

B1

RT UPLC = 0.96 min m/z 279 [M-H] –

λmax262 nm

O O

OH O

O O B8

-λ max 230, 281, 324, 337, 404 nm

O O

OH O

O O B8

λ max 230, 281, 324, 337, 404 nm

O O

OH O B3

-λmax284 nm

O O

OH O B3

λmax284 nm

O O

OH

-λ max 225, 284 nm

OH

λ max 225, 284 nm

B4

Octaketides

O O O O O OH O A7

-λ max 230, 280 nm

O O O

O O OH

O A4

-λmax284 nm

O O

OH

O A6

-λmax282 nm

CoAS O Acetyl-CoA

O O

OH

-λ max 270 nm

A1

O O

OH O A2

-λmax 285 nm O

O

OH O A8

-λmax309 nm

-λ max 283 nm

OH

O

O O A9

-λ max 243, 251, 292 nm O

O O

OH

O O A10

-λmax 235, 276 nm

Octaketides

O O O O O OH O A7

-λ max 230, 280 nm

O O O

O O OH

O A4

-λmax284 nm

Octaketides

O O O O O OH O A7

-λ max 230, 280 nm

O O O O O OH O A7

λ max 230, 280 nm

O O O

O O OH

O A4

-λmax284 nm

O O O

O O OH

O A4

RT UPLC = 0.84 min m/z 317 [M-H] –

λmax284 nm

O O

OH

O A6

-λmax282 nm

CoAS O Acetyl-CoA

O O

OH

-λ max 270 nm

A1

O O

OH O A2

-λmax 285 nm O

O

OH O A8

-λmax309 nm

-λ max 283 nm

OH

O

O O A9

-λ max 243, 251, 292 nm O

O O

OH

O O A10

-λmax 235, 276 nm

O O

OH

O A6

-λmax282 nm

O O

OH

O A6

λmax282 nm

CoAS O Acetyl-CoA

O O

OH

-λ max 270 nm

A1 O O

OH

λ max 270 nm

A1

O O

OH O A2

-λmax 285 nm

O O

OH O A2

λmax 285 nm O

O

OH O A8

-λmax309 nm

O

OH O A8

RT UPLC = 1.37 min m/z 191 [M-H] –

λmax309 nm

-λ max 283 nm

OH

λ max 283 nm

OH

O O

OH

O

O O A9

-λ max 243, 251, 292 nm

O

O O A9

λ max 243, 251, 292 nm O

O O

OH

O O A10

-λmax 235, 276 nm

O

O O

OH

O O A10

λmax 235, 276 nm

Fig 4 Structures of enzymatic reaction products of H perforatum HpPKS2 with different starter substrates The structures were deter-mined by UPLC ⁄ ESIMS.

Trang 6

Localization of HpPKS2 transcripts in

H perforatum tissues

In order to obtain more insight into the role of

HpPKS2 in H perforatum, in situ RNA hybridization

studies were performed Digoxigenin (DIG)-labelled

HpPKS2 RNA probes were used to hybridize

ﬁxed tissue sections of the leaves and ﬂower buds of

H perforatumin order to localize exactly the HpPKS2

transcripts in the tissues After hybridization of the

cross-sections of the leaves with a HpPKS2 RNA anti-sense probe, a dark blue signal that indicates HpPKS2 expression was clearly observed in the leaf margins (Fig 6A) The signal was speciﬁcally localized in the multicellular nodular structures between the lower epi-dermis and the photosynthetic parenchymal cells of the

H perforatum leaves Under test conditions, no signifi-cant background staining was observed, and the HpPKS2 probe specificity was confirmed by the absence of signal in the negative control sections of the leaves hybridized with HpPKS2 RNA sense probe (Fig 6B)

In the hybridized sections of the flower buds, a strong dark blue signal for HpPKS2 transcripts was localized in the petals (Fig 6C) and the stamens between anthers (Fig 6E), also restricted to multi-cellular nodules The nodules that showed the HpPKS2 expression in flower buds were structurally similar to those found to contain HpPKS2 transcripts in the leaf sections No signal was observed in the corresponding areas of the negative controls of the flower bud sections hybridized with HpPKS2 RNA sense probe (Fig 6D,F)

Multicellular nodules showing HpPKS2 expression

in both leaves and flower buds consisted of a core of large cells that was surrounded by one to three flat cell layers The HpPKS2 transcripts were found to be pres-ent in the large cells and also in the some of the inner-most flat cells of the nodules In the flower petals of

H perforatum, two types of multicellular nodules that share the same anatomical organization in the cross-sections have been reported previously [10] Spheroidal nodules similar to those observed in the leaf margins are also present in the petal margins, whereas the nod-ules in the interior parts of the petals are elongated tubulars [10,12] In this study, nodules in both the margins and the interior parts of the petals were found

to contain HpPKS2 transcripts

Localization of hypericins in H perforatum tissues

As reported previously [25], the leaf margins and ﬂower buds contain the highest amounts of hypericins

in H perforatum To see exactly where the red hyperic-ins are located, unstained cross-sections of the leaves and ﬂower buds of H perforatum were observed under

a microscope The dark red hypericins could be easily located because they remained in the parafﬁn sections and did not disappear until the in situ RNA hybridiza-tion Leaf cross-sections showed dark red material in multicellular nodules in the leaf margins (Fig 7A) The nodules were included between the lower

O

OH

R

-CO2

O O O H

O

H

m/z 191

O

H

O

OH

O

H

O

H

O

O H

-CO2

-CH2O

-CO 2

-CO2

or

O

H

O

OH

O

H

O H

O O OH

O H

O H OH

O

H

O

H

O O O H

O H

O O O

H

O

OH

R

OH

R

m/z 125

m/z 167

-CO2

O O O H

O

H

O

H

O

OH

O

H

O O O H

O

H

m/z 317

m/z 125

O

H

O

OH

O

H

O

H

O

O H O

O

H

O

O H

m/z 231 m/z 189

-CO2

-CH2O

-CO 2

-CO2

m/z 317 m/z 273

m/z 229

m/z 287 m/z 243

or

O

H

O

OH

O

H

O H

O O OH

O H

O H OH

O

H

O

H

O O O H

O H

O O O

H

A

B

C

D

Fig 5 MS fragmentation patterns of (A) a-pyrones, (B) SEK4 and

(C) SEK4b in the negative ionization mode, and (D) aloesone in the

positive ionization mode Fragment ions were identified based on

their exact masses.

Trang 7

epidermis and the photosynthetic parenchymal cells of

leaves, and they comprised a core of large cells

surrounded by ﬂat cell layers (Fig 7B) Red material

was present in the large cells and also in the some of

the innermost ﬂat cells of the nodules Dark red

multicellular nodules of the same structure were also observed in cross-sections of the ﬂower buds (Fig 7C) Smaller red nodules were present in the ﬂower petals (Fig 7D), whereas larger ones were found in the stamens between anthers (Fig 7E) The red material

Fig 6 In situ RNA localization of HpPKS2

transcripts in leaves and flower buds of

H perforatum Cross-section of (A) leaf, (C)

petal of flower bud and (E) stamen of flower

bud hybridized with DIG-labelled HpPKS2

RNA antisense probe (B,D,F) Corresponding

sections were hybridized with HpPKS2 RNA

sense probe Arrows point to multicellular

nodules Bars = 100 lm.

E

Fig 7 Localization of hypericins in leaves

and flower buds of H perforatum.

Unstained cross-sections of (A) leaf (B)

showing red pigmented nodules in leaf

margins and (C) flower bud (D) showing red

pigmented nodules in petal and (E) in

stamen Small arrows point to multicellular

nodules Bars = 100 lm.

Trang 8

was present in the nodules of both the margins and

the interior parts of the ﬂower petals

Discussion

Despite the fact that hypericins are pharmacologically

important compounds of H perforatum, a widely used

herbal remedy for the treatment of depression [1,2],

there is little information available about the

biosynthe-sis of these compounds To date, only one gene has been

cloned and characterized from the biosynthetic route

leading to hypericins The enzymatic product of hyp-1

has been shown to catalyse the ﬁnal stages of hypericin

biosynthesis [20] It has been proposed that type III

PKS would attend to the formation of emodin anthrone,

the initial key reaction step in the biosynthesis of

hypericins [20], but no such activity has been reported

In this study, the role of a newly found PKS from

H perforatum, HpPKS2 [25], was investigated for its

possible involvement in the biosynthesis of hypericins

Phylogenetic analysis showed that the plant-speciﬁc

type III PKS family proteins grouped into CHSs and

other functionally divergent PKSs (Fig 2) The only

exceptions were STSs from Fabaceae and

Gymno-sperms grouping with CHSs from the same or related

species STSs have been proposed to have evolved

independently from CHSs several times, which explains

their presence in several clusters in the phylogenetic

tree [31–33] HpPKS2 of H perforatum grouped

with functionally divergent non-chalcone-forming

plant-speciﬁc type III PKSs The grouping of HpPKS2

with non-CHSs indicates that HpPKS2 is not involved

in the biosynthesis of ﬂavonoids in H perforatum The

functionally divergent PKSs include, for example,

OKS and PCS from A arborescens, which accept

malonyl-CoA or acetyl-CoA as a starter substrate to

produce octaketides (SEK4 and SEK4b) and

pentake-tide chromone (5,7-dihydroxy-2-methyl-chromone),

respectively [26,27] However, HpPKS2 was not

partic-ularly closely related to any of the currently known

type III PKSs, which indicates that it is a novel

plant-speciﬁc type III PKS family protein We have

previously reported that the deduced amino acid

sequence of HpPKS2 shares only < 52% identity with

previously isolated type III PKSs [25]

HpPKS2 expressed in E coli resulted in an enzyme

of 43 kDa (Fig 3) The size coincides with a

predicted molecular mass of 43.1 kDa for HpPKS2,

calculated using bioinformatics tools [25], and with

that of a subunit size typical to plant-speciﬁc type III

PKSs The plant-speciﬁc type III PKSs are reported

to be homodimeric proteins with a subunit size of

40–45 kDa [21,23]

Functional characterization of the purified recombi-nant HpPKS2 revealed the expected OKS activity But instead of producing emodin anthrone, an octaketide precursor of hypericins, the enzyme catalysed the con-densation of one molecule of acetyl-CoA with seven molecules of malonyl-CoA to form unnatural octake-tides SEK4 and SEK4b (Fig 4) SEK4 and SEK4b, the longest polyketides known to be produced by type III PKSs, have also been shown to be the products of OKS from A arborescens [26] and shunt products of minimal type II PKS from Streptomyces coelicolor [34,35] The A arborescens OKS, along with HpPKS2, is the only enzyme among unmodified plant-specific type III PKSs that has been shown to have OKS activity Because the aloe does not accumulate SEK4 and SEK4b, the aloe OKS has been suggested to be involved in the biosynthesis of anthrones and anthraquinones in the plant and SEK4⁄ SEK4b produced in the absence of additional tailoring enzymes in vitro [26,36] The A arborescens OKS preferred malonyl-CoA as a starter substrate for the production of SEK4 and SEK4b Because SEK4 and SEK4b could not be found from incubations with starter substrates other than acetyl-CoA in this study,

it is likely that HpPKS2 used only acetyl-CoA as a starter substrate for production of SEK4 and SEK4b HpPKS2 also catalysed the formation of tri- to heptaketide products, using acetyl-CoA as a starter substrate (Fig 4) Triketide and tetraketide pyrones are often biosynthesized in vitro by PKSs when incu-bated with acetyl-CoA as a starter substrate [37–39] Penta- to octaketides are more rare products Two dif-ferent pentaketide products have previously been reported to be produced by unmodified plant-specific type III PKSs These are 5,7-dihydroxy-2-methylchro-mone produced by PCS from A arborescens [27] and a-pyrone by PKS from Plumbago indica [39] The pen-taketide chromone structure A8 has not previously been reported to be produced by plant-specific type III PKS The hexaketide a-pyrone A1 produced by HpPKS2 can be classified as a derailment product of type III PKS The structure is also the product

of P indica PKS, along with hexaketide phenylpyrone [39] Because the pyrones are not found in P indica tissues, it has been suggested that the PKS in vivo would be involved in the biosynthesis of naphthoqui-none plumbagin and the pyrones produced in vitro in the absence of accessory enzymes [39] Of the three heptaketides produced by HpPKS2 using acetyl-CoA

as a starter substrate, one was aloesone Aloesone has previously been reported as a product of aloesone synthase of Rheum palmatum [31], a plant known to be rich with chromones, napthalenes and anthraquinones

Trang 9

Aloesone was also the product of A arborescens OKS,

along with SEK4 and SEK4b, after a single amino

acid mutation, i.e replacement of glycine by alanine,

as in the case of aloesone synthase in the Gly207 site

[26] HpPKS2 has serine in the corresponding site To

our knowledge, the other two heptaketides produced

by HpPKS2, chromone A10 and phenylpyrone A6,

have not previously been reported as products of

plant-speciﬁc type III PKSs Notably, OKS and PCS

from A arborescens, PKS from P indica, aloesone

synthase from R palmatum and now HpPKS2 from

H perforatum all share mechanistically related

reac-tions, such as accepting acetyl-CoA⁄ malonyl-CoA as a

starter substrate, performing high numbers of

conden-sations and two to three cyclization reactions Because

most type III PKSs perform only one to three

exten-sions and catalyse the formation of one six-membered

ring, it can be assumed that the above-mentioned

PKSs may be involved in the biosynthesis of

structur-ally similar types of compounds in plants

The acceptance of other, larger starter substrates by

HpPKS2 shows that the enzyme has a broad substrate

acceptance, as reported for other type III PKSs

[22–24,26,27,32,37,39,40] HpPKS2 accepted both

aro-matic and aliphatic CoA-esters as starter units By

using isobutyryl-CoA, benzoyl-CoA and

hexanoyl-CoA as starter substrates, HpPKS2 produced tri- to

heptaketide products, mostly pyrones (Fig 4) In

addi-tion to pyrones, some chromones and phloroglucinols

were also produced It should be noted that with

star-ter substrates other than acetyl-CoA, HpPKS2 was not

able to produce octaketides but only afforded shorter

products supporting the view that acetyl-CoA could be

the real starter substrate for HpPKS2 in vivo

To our knowledge, the compounds produced by

HpPKS2 in vitro, which were mostly pyrones, have not

been described as constituents of H perforatum

Sev-eral recombinant plant-speciﬁc type III PKSs are

known to biosynthesize metabolites, especially pyrones,

that have not been described as being accumulated by

their plants of origin [26,32,37,39,40] The products

have been found to be typical for in vitro incubations

of type III PKSs with non-physiological substrates,

non-optimal assay conditions and are also suggested to

be produced in the absence of co-operating tailoring

enzymes [23,26,30,32,39] To date, the only

character-ized example of such a co-operating interaction of

plant-speciﬁc type III PKS with tailoring enzyme is the

biosynthesis of 6¢-deoxychalcone [23] Typically, type I

and type II PKSs consist of many additional subunits,

including ketoreductases, cyclases and aromatases, that

are often needed for the production of speciﬁc cyclized

polyketide products [34,35,41–43] These additional

subunits interact with PKS to stabilize the highly reac-tive polyketide chain preventing non-speciﬁc cycliza-tions It is not currently known whether emodin anthrone biosynthesis requires additional enzymes and thus it is possible that HpPKS2 failed to produce emo-din anthrone in this study because of the absence of additional tailoring enzymes in vitro

To further study the role of HpPKS2 in H perforatum,

in situ RNA hybridization studies to locate HpPKS2 transcripts were performed HpPKS2 expression was found to localize speciﬁcally in multicellular nodules in the leaf margins, ﬂower petals and stamens of H per-foratum (Fig 6) These types of structures present in the H perforatum tissues have been described previ-ously by several authors, and are referred to as dark glands [10,17,18,44] In this study, the same nodules were also found to contain dark red material (Fig 7) The red material in the dark glands has previously been found to consist of hypericins, and their accumu-lation is shown to be restricted to only the dark glands

in H perforatum [12,16–18] The obtained results are consistent with our previous study in which the expres-sion of HpPKS2, measured using real-time PCR, was shown to correlate with the concentrations of hyperic-ins in different H perforatum tissues [25] Recently, emodin, which is an oxidized derivative of emodin anthrone (Fig 1), has also been found to accumulate

at high concentrations in the dark glands of H perfo-ratum [12] The presence of emodin in only the dark glands in H perforatum suggests that emodin biosyn-thesis may take place in the glands [12] The restriction

of HpPKS2 expression and the presence of both hyper-icins and emodin speciﬁcally in the same cells imply that HpPKS2 may have a role in the biosynthesis of hypericins in H perforatum The localization of the HpPKS2 transcripts in the dark glands that accumu-late hypericins is very similar to the expression pattern

of type III PKS from Humulus lupulus Valerophenone synthase from H lupulus is responsible for the biosyn-thesis of the phloroglucinol skeleton of hop resin, and

it has been shown to be expressed speciﬁcally in secre-tory structures called ‘lupulin glands’ accumulating the resin [45]

The HpPKS2 transcripts were found to accumulate into both the large cells and some of the innermost ﬂat cells of the multicellular nodules This indicates that if HpPKS2 is involved in the biosynthesis of hypericins, then at least the early phase of biosynthesis, i.e the formation of emodin anthrone, may occur in both cell types Kornfeld et al [18] hypothesized that the biosynthesis of hypericins takes place in the peripheral ﬂat cells rather than in the large interior cells of the nodules

Trang 10

Based on these results, H perforatum HpPKS2 is a

novel plant-speciﬁc type III PKS having OKS activity

Furthermore, our ﬁndings show a strong connection

between HpPKS2 expression and the accumulation of

hypericins, indicating that HpPKS2 may have a role in

the initial key reaction step in the biosynthesis of

hypericins in H perforatum However, although the

enzyme is capable of carrying out the expected number

of condensation reactions in vitro, it fails in the

cyclization of the produced octaketide chain to emodin

anthrone The formation of derailment products by

HpPKS2 may mean that the biosynthesis of emodin

anthrone requires some additional, as yet unidentiﬁed

factors that are missing in vitro Recently, several

type III PKSs have been isolated that do not, in vitro,

produce the metabolites that they are expected to

cata-lyse and that are found in their plant of origin

There-fore, further studies are needed to elucidate the

reasons for these failures to reveal the actual

biosynthesis mechanism of many plant polyketides,

including hypericins

Experimental procedures

Construction of expression plasmid

described previously [25] The coding region of HpPKS2

was ampliﬁed from the cDNA by PCR, using forward

primer 5¢-CATATTGGGATCCATGGGTTCCCTTGAC-3¢

(the translation start codon is in bold and the BamHI site

is underlined) and reverse primer 5¢-ACGCTGGTACC

codon is in bold and the KpnI site is underlined) The PCR

was performed with DyNazyme II DNA polymerase

(Finnzymes, Espoo, Finland) The PCR conditions were

The ampliﬁed PCR product was puriﬁed by electrophoresis

PCR fragment of the expected size ( 1.2 kb) was excised

from the gel and further puriﬁed using Montage DNA

Gel Extraction Kit (Millipore, Bedford, MA, USA) The

puriﬁed PCR product was digested with BamHI and KpnI

pQE30 (Qiagen, Hilden, Germany) Thus, the recombinant

enzyme contains an additional hexahistidine tag at the

N-terminus The resulting recombinant plasmid pQE30–

Terminator Cycle Sequencing Kit (Applied Biosystems,

Foster City, CA, USA) and an ABI 310 DNA sequencer

(Model 377; Applied Biosystems)

Expression of recombinant HpPKS2

The recombinant plasmid pQE30–HpPKS2 was transferred into the E coli host strain M15 [pREP4] (Qiagen) for pro-tein expression E coli cells harbouring the plasmid were grown in Luria–Bertani liquid medium in the presence of

culture had been cooled on ice, IPTG (Roche, Basel, Swit-zerland) was added to the culture in a ﬁnal concentration

of 0.4 mm to induce protein expression The culture was

Enzyme purification

20 min) and resuspended in a lysis buffer (50 mm sodium phosphate buffer, pH 8.0, containing 500 mm NaCl, 10 mm b-mercaptoethanol, 1% Tween 20 and 20 mm imidazole)

sonication (Type UP50H; Dr Hielscher GmbH, Teltow, Germany) The lysate was diluted twofold with the same buffer and centrifuged at 17 000 g for 30 min The super-natant was collected for puriﬁcation of recombinant protein under native conditions according to the protocol of the QIAexpressionist [46], using Ni-NTA agarose Unbound proteins were washed away with a wash buffer (50 mm sodium phosphate buffer, pH 7.0, containing 500 mm

Tween 20 and 20 mm imidazole) and the recombinant protein was eluted with an elution buffer (50 mm sodium phosphate buffer, pH 7.0, containing 500 mm NaCl, 10 mm b-mercaptoethanol, 10% glycerol and 250 mm imidazole) After puriﬁcation, the protein concentration was deter-mined according to Bradford [47], using BSA (Sigma, St Louis, MO, USA) as a standard The purity of the protein

stacking gels The proteins were run along with protein markers (Fermentas, Vilnius, Lithuania) at 180 V, using a Mini-Protean II electrophoresis system (Bio-Rad, Hercules,

CA, USA) followed by staining with Coomassie Brilliant Blue R-250 (Merck, Darmstadt, Germany)

Polyketide synthase assays

Puriﬁed recombinant HpPKS2 (100 lg) was mixed with

200 lm starter substrates (acetyl-CoA, isobutyryl-CoA, ben-zoyl-CoA or hexanoyl-CoA; Sigma) and 300 lm malonyl-CoA (Sigma) An assay buffer (0.5 m potassium phosphate buffer, pH 6.8, containing 2.8 mm b-mercaptoethanol and

10 lm dithiothreitol) was then added to 500 lL For con-trol reactions, the enzyme was heat-denatured Incubations

stopped by adding 50 lL of 20% HCl, and the products were then extracted twice with 250 lL of ethyl acetate

Tiêu đề	Octaketide-producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins
Tác giả	Katja Karppinen, Juho Hokkanen, Sampo Mattila, Peter Neubauer, Anja Hohtola
Trường học	University of Oulu
Chuyên ngành	Plant biochemistry
Thể loại	Research article
Năm xuất bản	2008
Thành phố	Oulu, Finland

Định dạng
Số trang	14
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