In Scots pine and Norway spruce needles, flavonol 3-O-glucosides acylated at positions 3¢¢ and 6¢¢ with p-coumaric and ferulic acid are the main UV-B screen-ing pigments [24,25].. We hypo
Trang 1from Scots pine (Pinus sylvestris L.)
Florian Kaffarnik1,*, Werner Heller1, Norbert Hertkorn2and Heinrich Sandermann Jr1
1 Institute of Biochemical Plant Pathology, GSF-Research Center for Environment and Health, Neuherberg, Germany
2 Institute of Ecological Chemistry, GSF-Research Center for Environment and Health, Neuherberg, Germany
Several plant hydroxycinnamoyltransferases (HCTs)
have been described for the biosynthesis of
function-ally important secondary metabolites, e.g phytoalexins
[1–3] or flower pigments [4–7] They most commonly
use CoA esters as activated donor substrates [8] and
transfer the hydroxycinnamoyl moiety to a hydroxyl
or amino group of acceptor substrates Other donor
substrates such as glucosyl esters are occasionally
observed [9] Acceptor substrates include anthocyanin
glycosides [4–7,10], a flavonol 3-O-glycoside [11],
amines [1–3,12–15], meso-tartrate, shikimate and
qui-nate [16,17], fatty acids [18] or alkaloids [19] (for an
overview see [20]) The biochemistry of HCTs using
anthocyanin glycosides, or amines such as agmatine, tyramine or anthranilate, as acceptor substrates has been investigated in more detail [1,3,4,10,12,14,21] Genes encoding N-HCTs acting on amines such as tyr-amine, noradrenaline and serotonin [22,23] as well as O-HCTs acting on anthocyanins [6,7,10] have recently been cloned
In Scots pine and Norway spruce needles, flavonol 3-O-glucosides acylated at positions 3¢¢ and 6¢¢ with p-coumaric and ferulic acid are the main UV-B screen-ing pigments [24,25] We hypothesized that the final acylation that results in a dramatic absorption increase
of the molecules in the UV-B range (280–315 nm) [26]
Keywords
hydroxycinnamoyl-CoA flavonol
3-O-glycoside hydroxycinnamoyltransferases;
diacylated flavonol 3-O-glycosides; Scots
pine; Pinus sylvestris
Correspondence
W Heller, Institute of Biochemical Plant
Pathology, GSF-Research Center for
Environment and Health, D-85764
Neuherberg, Germany
Fax: +49 89 3187 3383
Tel: +49 89 3187 3041
E-mail: heller@gsf.de
Website: http://www.gsf.de/Forschung/
Institute/biop_intro.phtml
*Present address
Sainsbury Laboratory, John Innes Centre,
Norwich NR4 7UH, UK
(Received 24 November 2004, revised 14
January 2005, accepted 19 January 2005)
doi:10.1111/j.1742-4658.2005.04574.x
Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at posi-tions 3¢¢ and 6¢¢ are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles The last steps in the biosyn-thesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specifici-ties for positions 3¢¢, 4¢¢ or 6¢¢ The positions of the acyl groups were identi-fied by cochromatography with reference compounds and by NMR spectroscopy The enzymes were characterized by molecular mass, isoelec-tric point, and also pH and temperature optima Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3¢¢- and 6¢¢HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA In addition, acylation takes place in
a well-defined order, beginning at position 6¢¢ followed by acylation at posi-tion 3¢¢ These results give the first detailed characterizaposi-tion of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280–315 nm) radiation
Abbreviations
HCT, hydroxycinnamoyl-CoA flavonol 3-O-glucoside hydroxycinnamoyltransferase (EC 2.3.1.-); I3G, isorhamnetin 3-O-glucoside; K3G, kaempferol 3-O-glucoside; Q3G, quercetin 3-O-glucoside.
Trang 2is mediated by hydroxycinnamoyltransferase enzymes.
These steps would introduce p-coumaric and ferulic
acid residues at position 3¢¢ and ⁄ or 6¢¢, respectively,
of flavonol 3-O-glucosides In the Scots pine,
3-O-glycosides of three different flavonol types, namely
kaempferol, isorhamnetin and quercetin, have been
detected Interestingly, similar diacylated flavonol
3-O-glycosides are not only found in coniferous leaves but
also in the leaves of broadleaf trees, such as oak
spe-cies [27,29] This suggests that these metabolites may
play an important role in UV-B screening in a variety
of economically important tree species The objective
of this study was to provide biochemical information
on HCTs as a basis to understand the mechanisms
of biosynthesis of UV-B screening pigments in the
Scots pine In this paper, HCTs acylating flavonol
3-O-glucosides are biochemically characterized in more
detail for the first time
Results and Discussion
Detection of HCT activities in cell extracts from
Scots pine needles
Cell extracts from developing needles of Scots pine
trees were assayed for the presence of HCT activities
Kaempferol, quercetin and isorhamnetin
3-O-gluco-sides (K3G, Q3G and I3G, respectively; Fig 1) as well
as the monoacylated 6¢¢-p-coumaroyl K3G (tiliroside)
were tested as acceptors with p-coumaroyl-CoA as the
acyl donor Incubation of crude cell extracts with I3G
and p-coumaroyl-CoA, and analysis of the products
with HPLC led to four different products that were
recognized as acylated compounds by their UV spectra
(Fig 2A, upper panel, peaks 1 through 4, and 2B)
The other peaks of Fig 2A gave UV-spectra typical
Fig 1 Structure of flavonol 3-O-glucosides In Scots pine needles
three different acylated flavonol 3-O-glucosides are found (R: H,
kaempferol; OH, quercetin; OMe, isorhamnetin) These compounds
are acylated at position 3¢¢ with p-coumaric acid and at position 6¢¢
with either p-coumaric acid or ferulic acid.
A
B
Fig 2 HPLC analysis of enzyme products Crude cell extracts from Scots pine needles were assayed with isorhamnetin 3-O-glucoside (A, upper panel; I3G) and 6¢¢-p-coumaroyl-kaempferol 3-O-glucoside (A, lower panel; tiliroside) as the acceptor and p-coumaroyl-CoA as the donor substrates I3G was chosen as the nonacylated substrate with crude cell extracts because the respective product 1 with K3G comigrated with a minor nonflavonoid hydroxycinnamoyl by-product which prevented quantification of 6¢¢-p-coumaroyl-kaempferol 3-O-glucoside S, substrate; IS, internal standard; pinosylvin methyl ether.The peaks marked 1–6 were identified as acylated products
by their diode array spectra (B) Characteristics of the UV spectra
of acylated compounds are the absorption maximum at 315 nm due to the hydroxycinnamic acid moieties and shoulders at 270 and
350 nm originating from the flavonol 3-O-glycoside [26] Differences between monoacylated (1–3) and diacylated compounds (4–6) con-sist in a higher proportion of the absorbances at 315 nm and
350 nm for diacylated compared to monoacylated compounds Spectra shown are normalized at 315 nm.
Trang 3for simple nonflavonoid p-coumaric acid derivatives.
K3G and I3G gave similar results (data not shown)
but one of the p-coumaric acid related by-products
detected in the assays with I3G as substrate
comigrat-ed with tiliroside, one of the reaction products of K3G
(Fig 2A) Using tiliroside as the acceptor substrate
one minor and one major diacylated product were
detected (Fig 2A, lower panel, peak 5 and 6) In
con-trol assays with heat-inactivated enzyme preparations,
or omitting either donor or acceptor substrate, none of
the expected acylated products was obtained Diode
array spectra (Fig 2B) showed similar absorption
pat-terns for compounds 1–3 with absorption ratios of
approximately 1.9 between 316 and 350 nm These
spectra differed slightly from those of compounds 4–6,
which showed a shoulder of markedly lower intensity
at 350 nm and absorption ratios of approximately 2.9
This is in good agreement with ratios of 1.6 and 2.5
measured for tiliroside and 3¢¢,6¢¢-di-p-coumaroyl K3G,
respectively (data not shown), and can be explained
by the higher absorption at 315 nm of diacylated
compared to monoacylated metabolites [26,28]
Com-pounds 1–3 thus appeared to be monoacylated, and
compounds 4–6 diacylated, products Furthermore,
compound 4 exhibited a comparable retention time
and absorption pattern to compound 6, suggesting the
same acylation pattern in both compounds The
retent-ion times of compound 1 and tiliroside (Fig 2A, ‘S’ in
lower panel) were also similar, indicating the same
acy-lation position for both compounds A small shift to
longer retention time of compounds 1 and 4, relative
to tiliroside and compound 6, respectively, was appar-ently caused by the slightly higher lipophilicity of the isorhamnetin relative to the kaempferol derivatives, owing to the additional methoxy function of isorham-netin The agreement of the acylation pattern of com-pound 1 with that of tiliroside was further confirmed
by coinjection experiments with tiliroside and com-pound 1 derived from enzyme assays with K3G as sub-strate (data not shown)
Taken together, our results show that at least three different monoacylated and two diacylated products of flavonol 3-O-glucosides were formed by HCT activities
in crude cell extracts from Scots pine needles Coinjec-tion experiments of authentic 6¢¢-p-coumaroyl K3G standard (tiliroside) allowed the identification of the respective enzyme product observed in the chromato-grams
Separation of HCT activities The formation of several products in assays of crude cell extracts raised the question of whether different enzymes were involved The separation of enzyme activities was successfully carried out by anion exchange chromatography of protein on Q-Sepharose after ammonium sulfate precipitation The fractions eluting from the ion exchange column were tested with both K3G and tiliroside as substrates Three separate activities were detected with K3G (Fig 3A; peaks I–III) Peak I represents the protein fraction not retained by the column and gave a product
corres-A
B
Fig 3 Separation of hydroxycinnamoyltransferase activities by anion exchange chromatography on Q-Sepharose Protein extracted from Scots pine needles was chromatographed on Q-Sepharose after ammonium sulfate precipitation HCT activities of collected fractions were determined with K3G (A) and tiliroside (B) as substrates Using K3G three different HCT activities (A, I–III) were separated giving products that corresponded to compound 1 (d), compound 2 ( ) and compound 3 (.) in Fig 2A In contrast, only two activities were detected with tiliroside (B, IV and V) giving compounds 5 ( ) and 6 (.), respectively, in Fig 2A The solid line represents protein concentration, measured
as absorption at 280 nm The dotted line shows changes in conductivity, caused by the sodium chloride gradient applied.
Trang 4ponding to compound 1 in Fig 2A The products of
peaks II and III eluting upon application of an NaCl
concentration gradient corresponded respectively to
compounds 2 and 3 in Fig 2A Peaks IV and V were
detected with tiliroside (Fig 3B), and corresponded to
peaks II and III in Fig 3A, and the products were
compounds 5 and 6, respectively in Fig 2A In the
protein fraction that was not retained by the column,
no activity was detectable with tiliroside as the
sub-strate (Fig 3B) This supported the above finding that
compound 1 corresponds to tiliroside, which is already
acylated at position 6¢¢ Thus, it was concluded that
three position-specific HCTs exist in Scots pine
needles, and the activity that was not bound to the
anion exchange matrix can be assigned to 6¢¢HCT
Identification of products from HCT reactions
The positional specificity of the two as yet unassigned
HCTs was determined via spectroscopic identification
of compounds 2 and 3 enzymatically prepared by
incu-bations using appropriate enzyme fractions eluted from
anion exchange chromatography Incubations were
performed with K3G and p-coumaroyl-CoA as
sub-strates, and the products were purified by preparative
HPLC and analyzed by 1D-1H-NMR and 2D-1H-1
H-COSY-NMR spectroscopy
The product corresponding to compound 2 of Fig 2
showed a chemical shift for H-4¢¢ of 4.79 p.p.m
com-pared to 3.23 p.p.m of the nonacylated K3G,
indica-ting that the acyl group was at position 4 of the
glucose molecule On the other hand, the product
cor-responding to compound 3 of Fig 2 showed a
chem-ical shift for H-3¢¢ of 5.02 p.p.m compared to
3.34 p.p.m of K3G, and was therefore acylated at
position 3 of the glucose molecule The NMR data
(see Experimental procedures for details) combined
with the results of cochromatography thus proved the
existence of three separate position-specific enzymes,
i.e 3¢¢-, 4¢¢- and 6¢¢HCT, in Scots pine needles Both
3¢¢- and 4¢¢HCT convert nonacylated flavonol
3-O-glucosides in addition to the 6¢¢-monoacylated
tiliroside, giving the respective monoacylated 3¢¢- and
4¢¢-p-coumaroyl flavonol 3-O-glucoside (Fig 2;
com-pounds 3 and 2), and diacylated 3¢¢,6¢¢- and
4¢¢,6¢¢-di-p-coumaroyl K3G (Fig 2; compounds 6 and 5) The
simultaneous presence of 3¢¢HCT and 6¢¢HCT in crude
cell extracts directly gave rise to diacylated products of
flavonol 3-O-glucosides, e.g compound 4 in Fig 2
Consistently, this is in agreement with the acylation
pattern found in Scots pine, where p-coumaric and
ferulic acids were identified at positions 3¢¢ and 6¢¢ of
flavonol 3-O-glucosides [28] The discovery of products
acylated at position 4¢¢ was somewhat surprising, because no corresponding metabolites have been des-cribed from Scots pine so far However, the occurrence
of only low 4¢¢HCT activities in crude cell extracts of Scots pine needles indicate that flavonol 3-O-glycosides acylated at position 4¢¢ may be present as minor com-pounds that have not been recognized yet in this plant
On the other hand, metabolites with this structural fea-ture have earlier been identified from leaves of ever-green Quercus species [27,29]
General properties of the HCT enzymes
We investigated the biochemical properties of the par-tially purified and separated enzyme activities after anion exchange chromatography (Fig 3) The appar-ent molecular masses as determined with a Superose 6 column were 47 ± 2 kDa for 3¢¢HCT and 35 ±
3 kDa for 4¢¢HCT The value for enzymatically active 6¢¢HCT was only 9 kDa under the same conditions This surprisingly low value was attributed either to interaction of the protein with the gel matrix or to action of proteases during purification Therefore, an ammonium sulfate fraction was prepared in the pres-ence of protease inhibitors, and chromatography was performed on a Superdex 75 column The apparent molecular mass of 6¢¢HCT now observed was
42 ± 3 kDa, while the values of the other two activit-ies were not altered Molecular mass data of acyl-transferases reviewed in [20] generally ranged between
40 and 70 kDa In the case of a trimeric quinate O-hydroxycinnamoyltransferase, however, a value as low as 15 kDa for the monomer was described [30] Partial proteolysis has been mentioned for some other acyltransferases [3]
Isoelectric points of the partially purified proteins were determined by chromatofocusing on a Mono-P column Both 3¢¢- and 4¢¢HCT had a pI of 4.7, whereas 6¢¢HCT appeared at pI 7.9 Maximal activities were determined for both 4¢¢- and 6¢¢HCT at pH 8 and
44C Half maximal values for 4¢¢HCT were obtained
at pH 6.8 and 8.5, and at 36 and 50C For 6¢¢HCT, half maximal values were at pH 6.5 and 9.2, and at 36 and 52C Maximal activity for 3¢¢HCT was at pH 7 and 40C and half maximal values were at pH 6.2 and 8.0, and at 28 and 47C
Kinetic parameters of 3¢¢- and 6¢¢HCT Partially purified 3¢¢- and 6¢¢HCT, the two major HCT activities in Scots pine needles, were tested for their kinetic parameters with p-coumaroyl- and feru-loyl-CoA as donor substrates and K3G, tiliroside
Trang 5and 3¢¢-p-coumaroyl K3G as acceptor substrates
(Table 1) Using enzyme preparations after anion
exchange chromatography on Q-Sepharose 3¢¢HCT
showed a distinctly lower apparent Km value with
p-coumaroyl-CoA than with feruloyl-CoA, whereas
6¢¢HCT has comparable apparent Km values for both
CoA esters This is consistent with the observation
that the natural flavonol glycoside metabolites are
substituted at position 3¢¢ with p-coumaric acid, but
at position 6¢¢ with either p-coumaric acid or ferulic
acid [25,28]
Regarding the flavonoid substrate 3¢¢HCT showed a lower apparent Km value for tiliroside than for K3G, indicating a higher affinity towards the monoacylated compared with the nonacylated substrate Addition-ally, the ratio between Vmax and Km was clearly in favour of tiliroside as the natural substrate of 3¢¢HCT Furthermore, the apparent Km value of 6¢¢HCT for K3G was in the same range as the one of 3¢¢HCT for tiliroside while no activity of the 6¢¢HCT with 3¢¢-p-coumaroyl K3G was detected (Table 1) These find-ings result in a sequential acylation first at position 6¢¢ with p-coumaric or ferulic acid, followed by acylation
at position 3¢¢ only with p-coumaric acid as shown in Fig 4 This model reflects the natural occurrence of the respective metabolites [28] However, it cannot be excluded that other factors such as compartmentation
or metabolic channeling may contribute to or deter-mine the specificity of the substitution pattern in vivo [31]
Substrate specificity
To test the substrate specificity of 3¢¢- and 6¢¢HCT,
a number of flavonol 3-O-glycosides were analysed (Table 2) Variation of the B-ring substitution pattern
of the flavonol had minor but distinct influence on the transferase activities 3¢¢HCT showed higher activity with kaempferol and isorhamnetin 3-O-glucosides with a more lipophilic B-ring compared to quercetin
Table 1 Apparent Michaelis–Menten parameters of 3¢¢- and
6¢¢HCT The apparent Michaelis–Menten parameters were
deter-mined using enzyme preparations from anion exchange
chromato-graphy on Q-Sepharose which fully separated the HCT activities
(Fig 3) n.d., not detectable.
Enzyme Substrate
Km (l M )
Vmax (lkatÆkg)1)
Vmax⁄ K m (katÆ M )1Ækg)1)
3¢¢-p-Coumaroyl K3G b n.d n.d n.d.
a 100 l M tiliroside as fixed substrate b 100 l M p-coumaroyl-CoA as
fixed substrate c 100 l M K3G as fixed substrate.
Fig 4 Suggested sequential acylation of
flavonol 3-O-glucosides The K m values of
3¢¢HCT indicated a higher affinity to tiliroside
(16 l M ) than to K3G (47 l M ) While 6¢¢HCT
did not acylate 3¢¢-monocoumaroylated K3G
at position 6¢¢, the K m value for K3G (22 l M )
was in the same range as that of 3¢¢HCT for
tiliroside This indicates a sequential
acyla-tion of flavonol 3-O-glucosides, first at
posi-tion 6¢ followed by acylaposi-tion at posiposi-tion 3¢¢.
C, p-coumaroyl; F, feruloyl.
Trang 63-O-glucoside In contrast, 6¢¢HCT preferred a more
polar B-ring of the substrate showing the highest
activ-ity with quercetin 3-O-glucoside
Comparing different quercetin 3-O-glycosides
revealed high specificity towards glucose for both
enzymes (Table 2) For 3¢¢HCT the hydroxyl group at
position 4¢¢ clearly influences activity The
3-O-b-d-gal-actoside with axial configuration exhibited only 18%
activity under standard assay conditions compared to
the 3-O-glucoside with equatorial configuration On
the other hand, the presence or absence of the
hydroxymethyl group at position 5¢¢ has no major
effect indicated by comparable activities for the
3-O-b-d-galactoside and the
3-O-a-l-arabinopyrano-side For 6¢¢HCT the positions of 3¢¢ and 4¢¢ hydroxyl
groups are less important indicated by about half the
activity for the 3-O-b-d-galactoside compared to the
3-O-b-d-glucoside The 6¢¢desoxyglycoside quercetin
3-O-a-l-rhamnoside, which deviates particularly in
configuration at position 5¢¢, did not serve as a
sub-strate for 3¢¢HCT On the other hand, flavonoid
6¢¢des-oxyglycosides with d-configuration are not naturally
occurring in plants and were therefore not tested
Anthocyanin substrates, such as cyanidin
3-O-glu-coside, cyanidin 3,5-di-O-glucoside and cyanidin
3,2¢-di-O-glucoside were not transformed by both
3¢¢- and 6¢¢HCT under these conditions Anthocyanin HCTs have been shown to be active under comparable conditions [4–7,10]
In conclusion, based on the flavonol 3-O-glycoside specificity of HCTs described here, these key enzymes for the biosynthesis of UV-B screening pigments in the Scots pine may represent a separate functional group
of acyltransferases
Experimental procedures
Reference substances and substrates Flavonol 3-O-glycosides, as well as 6¢¢-p-coumaroyl-kaempf-erol 3-O-glucoside (tiliroside) were from Extrasynthe`se (Lyon, France) CoA esters of p-coumaric and ferulic acids were essentially synthesized according to a published method [32] The products (0.12 mmol) were purified using a Fracto-gel EMD DEAE 650 (S) column (Fracto-gel bed 12 mL) (Merck, Darmstadt, Germany) and an A¨KTA Explorer system (Amersham Biosciences, Freiburg, Germany) The solvents used were 0.1 m formic acid (A) and 1.5 m sodium formate (B) After application of the crude reaction product ( 0.25 mmol in 10 mL) the column was washed with 50 mL
of solvent A A gradient from 0 to 100% B in a total volume
of 110 mL was then applied, followed by 320 mL solvent B Fractions showing appropriate UV spectra (maxima at 259 and 334 nm for p-coumaroyl-CoA, 256 and 346 nm for feru-loyl-CoA) were collected, pooled and desalted on a Dowex
50 WX 8 column (Aldrich, Steinheim, Germany) Other chemicals used were of highest available purity and were pur-chased from Sigma (Steinheim, Germany)
Protein determination Protein concentration was measured according to the method of Bradford [33] using bovine serum albumin (BSA) as standard
Protein extraction Analytical scale Approximately 100 mg of needle material from seedlings
or pine trees, shock frozen in liquid nitrogen, was coarsely homogenized with pestle and mortar Fifty milligrams poly(vinylpolypyrrolidone) (PVPP) and 3 mg Celite were then added, and protein was extracted by further homoge-nization with three portions of 0.5 mL extraction buffer [100 mm sodium phosphate, 10% (w⁄ v) sucrose, 1.5% (w⁄ v) PEG 1450, 5 mm 1,4-dithioerythritol (DTE), pH 6.8]
in an ice bath [34] After two centrifugations (20 000 g,
4C, 5 min each) the supernatant was desalted on a NAP-5 column (Amersham Biosciences) according to the manufacturer’s instructions
Table 2 Comparison of relative activities of different flavonol
3-O-glycosides Relative enzyme activities were determined using
enzyme preparations from anion exchange chromatography on
Q-Sepharose which fully separated the HCT activities (Fig 3).
p-Coumaroyl-CoA was the donor substrate for all measurements,
and substrate concentrations of 100 l M were used for all
sub-strates.
Flavonol substrate
Substituent
at position 3¢
Relative activity (%) 3¢¢HCT 6¢¢HCT Flavonol 3-O-b- D -glucopyranosides
Kaempferol 3-O-Glc
(astragalin)
Quercetin 3-O-Glc
(isoquercitrin)
Quercetin 3-O-glycosides
Quercetin 3-O-b- D -glucopyranoside
(isoquercitrin)
Quercetin 3-O-b- D -galactopyranoside
(hyperoside)
Quercetin 3-O-a- L -arabinopyranoside
(guaijaverin)
Quercetin 3-O-a- L -rhamnopyranoside
(quercitrin)
Trang 7Preparative scale
Approximately 1700 g of needle material was harvested
from field-grown trees at the time of highest specific activity
(June and July), immediately frozen in liquid nitrogen and
ground with a pestle and mortar After lyophilization for
48 h, the dried material was ground for 3 min at 4C in an
analysis mill A10 (IKA Labortechnik, Staufen, Germany)
and stored at )80 C Cell extracts were prepared on ice
from 25 to 30 g needle powder, 60 g PVPP and 4 g Celite
in 100 mm sodium phosphate buffer, pH 6.8 containing
10% (w⁄ v) sucrose, 1.5% (w ⁄ v) PEG 1450, 1 mm DTE and
1 mm EDTA on ice [28] The extraction was followed by
two centrifugation steps at 30 000 g for 10 min at 4C In
some experiments, Complete Protease Inhibitor cocktail
(one tablet per 50 mL; Roche, Mannheim, Germany) was
included
Enzyme assays
Crude cell extracts
Enzyme assays were performed in a total volume of 212 lL
with 200 lL extract at protein concentrations between 50
and 100 lgÆmL)1and 6 lL each of hydroxycinnamoyl-CoA
(3.5 mm in H2O) and flavonol 3-O-glucoside (3.5 mm in
methanol) at final concentrations of 0.1 mm The reaction
was started by the addition of one of the substrates After
incubation at 37C for 60 min 1 nmol pinosylvin methyl
ether (0.177 mm in methanol) was added as internal
stand-ard, and the products were extracted with two portions of
200 lL ethyl acetate The organic phases were pooled and
dried under a stream of N2at room temperature The
resi-due was redissolved in 80 lL 50% (v⁄ v) acetonitrile in
H2O, and analyzed by HPLC after centrifugation at
20 000 g for 5 min
Partially purified fractions
The total assay volume was 100 lL in 100 mm sodium
phosphate, 5 mm DTE, pH 6.8 The final substrate
concen-trations and test procedure were as described above
Protein concentration and desalting
All steps were carried out at 4C or on ice The crude
cell extract was fractionated by ammonium sulfate
pre-cipitation (25–60% saturation) After centrifugation at
30 000 g for 30 min, an upper layer was formed,
contain-ing the protein and PEG 1450 [35] The
protein–PEG-phase was separated by filtration through Miracloth and
dilution into buffer A [20 mm Tris⁄ HCl buffer, pH 7.5
containing 10% (v⁄ v) glycerol, 1 mm DTE and 1 mm
EDTA] Desalting was performed using Sephadex G-25
(Amersham Biosciences)
Anion exchange chromatography
A 64 mL Q-Sepharose fast flow column (Amersham Bio-sciences) was pre-equilibrated with buffer A The concentra-ted extract (190 mL) was loaded onto the column, and after washing with two column volumes of the same buffer, the enzyme was eluted with a gradient from 0 to 0.5 m NaCl in five column volumes at a flow rate of 7.5 mLÆmin)1 Fractions of 10 mL were collected and assayed for HCT activity and protein concentration
Gel filtration chromatography
A Superose 6 HR 10⁄ 30 column (Amersham Biosciences) was pre-equilibrated with a buffer containing 100 mm sodium phosphate, pH 6.8, 100 mm NaCl, 10% (v⁄ v) gly-cerol and 1 mm DTE Fractions from the Q-Sepharose col-umn were pooled and concentrated using Centricon YM-10 filtration units (Millipore, Eschborn, Germany) Volumes of
100 lL protein solution were applied to the column and eluted at a flow rate of 250 lLÆmin)1 Fractions of 200 lL were collected and assayed for HCT activity and protein concentration The column was previously calibrated with the following molecular mass markers: b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carboan-hydrase (29 kDa) and cytochrome c (12.4 kDa) For deter-mination of the apparent molecular mass of the 6¢¢HCT after using protease inhibitors a Superdex 75 HR 10⁄ 30 (Amersham Biosciences) column was pre-equilibrated with
a buffer containing 100 mm sodium phosphate, pH 8.0,
100 mm NaCl, 10% (v⁄ v) glycerol, 1 mm DTE and Com-plete Protease Inhibitor cocktail (one tablet per 50 mL) A volume of 250 lL of a desalted ammonium sulfate fraction from a cell extract prepared from young needles in the pres-ence of Complete Protease Inhibitor cocktail was applied to the column, and fractions of 200 lL were collected and assayed for HCT activity The column was calibrated using BSA (66 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), myoglobin (17.6 kDa) and ribonuclease A (13.7 kDa) as standards
Chromatofocusing
A Mono-P HR 5⁄ 20 column (Amersham Biosciences) was pre-equilibrated with 25 mm Piperazine⁄ HCl (pH 5.2), 10% (v⁄ v) glycerol and 1 mm DTE or 25 mm diethanolam-ine⁄ HCl (pH 9.5), 10% (v ⁄ v) glycerol and 1 mm DTE for determination of the isoelectric point of 3¢¢- and 4¢¢HCT or 6¢¢HCT, respectively The pH gradient was generated in the column during the passage of a solution of Polybuffer 74 (1 : 10, pH 4.0) or Polybuffer 96 (1 : 10, pH 6.0) with 10% (v⁄ v) glycerol and 1 mm DTE The flow rate was 0.5 mLÆmin)1, and fractions of 0.5 or 0.8 mL were collected and assayed for HCT activity and protein concentration
Trang 8Enzyme characterization
The characterization of HCT activities was performed with
partially purified enzyme preparations after anion
exchange chromatography All measurements were
per-formed as triplicates For determination of
pH-depend-ence, enzyme preparations were buffer-exchanged with
NAP-5 columns (Amersham Biosciences) in 100 mm
sodium phosphate, pH 6.5–8.5 (3¢¢- and 4¢¢HCT) or
100 mm sodium phosphate, pH 6.0–8.0 and 50 mm
Tris⁄ HCl, pH 7.0–9.5 (6¢¢HCT) For determination of the
kinetic parameters Km and Vmax the following substrate
concentrations were used: p-coumaroyl- and feruloyl-CoA
10–450 lm with fixed acceptor concentrations of 100 lm,
kaempferol 3-O-glucoside 10–450 lm and tiliroside
10–350 lm with fixed donor concentrations of 100 lm
Calculation of the kinetic parameters was performed by
approximation of the received data to a Michaelis–Menten
function with sigma plot (Jandel Scientific, San Rafael,
CA, USA)
NMR analysis
For structure determination of enzyme products by NMR
analysis, compounds were synthezised enzymatically with
suitable protein fractions after anion exchange
chromato-graphy, using kaempferol 3-O-glucoside and
p-coumaroyl-CoA as substrates The enzyme assay was analogous to the
standard enzyme assay, but was upgraded to a volume of
2.0 mL, and an incubation time of 100 min A total of 90
assays (180 mL) was extracted with four portions of
120 mL ethyl acetate The organic phases were pooled and
dried in vacuo Products were purified with a preparative
HPLC system, consisting of a pump 114M, a controller
420, a system organizer 340, a detector 165 (all Beckman,
Mu¨nchen, Germany) and an integrator C-R3A
Chromato-pac (Shimadzu, Duisburg, Germany) Separation was
per-formed on a 250· 8.0 mm Spherisorb ODS2 5.0 lm
column (Bischoff, Leonberg, Germany) starting with 2 min
20% (v⁄ v) acetonitrile in water, followed by a gradient up
to 50% (v⁄ v) acetonitrile within 15 min and 3 min 50%
(v⁄ v) acetonitrile at 2.8 mLÆmin)1 Detection was performed
at 314 nm Appropriate peaks were manually collected and
identified by analytical HPLC For comparison, K3G,
til-iroside and 2¢¢,6¢¢p-di-coumaroyl kaempferol 3-O-glucoside
were measured as reference substances 1H NMR spectra
were acquired with a Bruker DMX 500 NMR spectrometer
(Rheinstetten, Germany) operating at 500.13 MHz proton
frequency from a few mg of sample in 750 lL CD3CN
(d1H¼ 1.93 p.p.m.) usually at 303 K with 90 deg pulses
[90(1
H)¼ 9.3 ls], acquisition time of 3.2 s and a
relaxa-tion delay of 7 s Gradient enhanced (length, 1 ms;
recov-ery, 450 ls), absolute value 2Q-COSY NMR spectra were
acquired with aq¼ 234 ms and 470 increments in F1 at a
sweep width of 4370 Hz
4¢¢-p-coumaroyl kaempferol 3-O-glucoside (analogue
to compound 2 in Fig 2)
1H-NMR (500 MHz, CD3CN, 273 K, c 150 lg): d¼ 8.09 (2H, AA¢; H-2¢ ⁄ 6¢), d ¼ 7.64 (H, d; H-7¢¢¢), d ¼ 7.50 (2H, AA¢; H-2¢¢¢ ⁄ 6¢¢¢), d ¼ 6.94 (2H, XX¢; H-3¢ ⁄ 5¢), d ¼ 6.82 (2H, XX¢; H-3¢¢¢ ⁄ 5¢¢¢), d ¼ 6.47 (H, d; H-8), d ¼ 6.32 (H, d; H-8¢¢¢), d ¼ 6.25 (H, d; H-6), d ¼ 5.21 (H, d; H-1¢¢), d ¼ 4,79 (H, t; H-4¢¢), d ¼ 3.64 (H, t; H-3¢¢), d ¼ 3.48 (H, dd; H-2¢¢), d ¼ 3.37 (H, dddd; H-5¢¢), d ¼ 3.30 (2H, m; H-6¢¢A, H-6¢¢B)
3¢¢-p-coumaroyl kaempferol 3-O-glucoside (analogue
to compound 3 in Fig 2)
1
H-NMR (500 MHz, CD3CN, 303 K, c 350 lg): d¼ 8.08 (2H, AA¢; H-2¢ ⁄ 6¢), d ¼ 7.70 (H, d; H-7¢¢¢), d ¼ 7.53 (2H, AA¢; H-2¢¢¢ ⁄ 6¢¢¢), d ¼ 6.95 (2H, XX¢; H-3¢ ⁄ 5¢), d ¼ 6.86 (2H, XX¢; H-3¢¢¢ ⁄ 5¢¢¢), d ¼ 6.51 (H, d; H-8), d ¼ 6.39 (H, d; H-8¢¢¢), d ¼ 6.29 (H, d; H-6), d ¼ 5.17 (H, d; H-1¢¢), d ¼ 5.02 (H, t; H-3¢¢), d ¼ 3.60 (H, t; H-2¢¢), d ¼ 3.54 (H, m; H-4¢¢), d ¼ 3.48 (H, m; H-6¢¢A), d ¼ 3.43 (H, dd; H-6¢¢B),
d¼ 3.28 (H, ddd; H-5¢¢)
HPLC analysis Analysis of enzyme products HPLC separation was performed according to [24] with the following modifications: a 250· 4.6 mm Spherisorb ODS2 5.0 lm column was run for 3 min with solvent A [1.9% (v⁄ v) formic acid, 0.1% (w ⁄ v) ammonium formate in water] followed by a gradient for 7 min to 35% solvent B [1.9% (v⁄ v) formic acid, 0.1% (w ⁄ v) ammonium formate, 9.6% (v⁄ v) water in acetonitrile], 7 min to 44% B, 5 min to 79%
B and 1 min to 100% B, detection was performed at
314 nm
Acknowledgements
We thank Susanne Stich for excellent technical assist-ance and Giovanni Romussi, Genova, for providing a sample of 2¢¢,6¢¢-di-p-coumaroyl kaempferol 3-O-glu-coside
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