Unexpected role of glutathione inthe oxidative transformation of flavan-3-ols by anthocyanidin synthase from Vitis vinifera

Jean Chaudière, Jia-Rong Zhang and Claudine Trossat-Magnin

ANS reactions

Anthocyanidins

unstable

ANS reactions

ANS from Arabidopsis thaliana, Perilla frutescens and Ginkgo biloba were not found to be

efficient producers of anthocyanidins in vitro.

ANS from Arabidopsis t. has in fact been reported to catalyze the oxidative transformation of

other polyphenols in vitro, such as naringenin, dihydroquercetin (DHQ) and catechin.

Anthocyanidins

unstable?

Generic substrate hydroxylationby iron-oxoglutarate dioxygenases

1. Production of free enzyme without tag

➢ E.coli strain: BL21(DE3) - Expression plasmid: pHGGWA

➢ Purification of the fusion protein by IMAC (Nickel-affinity column)

➢ Cleavage of the tag by thrombin

➢ Removal of the tag by IMAC (Nickel-affinity column)

➢ Removal of thrombin with p-Aminobenzamidine agarose

His6-GST-ANS

His6-GST

non-tagged ANS

His₆-GST tag ANSThrombin

Recognitionsite

Correct (expected) molecular mass

C-terminalN-terminal

Typical reaction conditionsUnblesse otherwise mentioned

➢ 2-Oxoglutarate 1 mM, Ascorbate 2 mM, NaCl 20 mM

➢ Fe2SO4 10 µM

➢ Catalase 200 U/mL

➢ MES buffer 20 mM, pH 6,5; 35oC (or Ammonium acetate buffer)

➢ VvANS close to 1 µM

➢ Polyphenol 50 or 100 µM

➢ Total volume = 20 mL

➢ Strong stirring (O2 not limiting)

➢ Reaction trigerred by polyphenol addition

➢ Analysis of medium after 30 min

➢ Systematic enzyme blanks

The Oxidative transformation of leucocyanidin by VvANS leads only to quercetin

The oxidative transformation of leucocyanidin leads only to quercetin

Zhang et al., J. Agric.Food Chem. 2018, 66, 351-358

Zhang et al., J. Agric.Food Chem. 2019, 67, 3595-3604

The first generic catalytic step of ANS is a C3-hydroxylation

which produces a 3,3-gem-diol

FinalProduct(s)

The Oxidative transformation of dihydroflavonols

leads only to flavonols

Dihydroflavonol

Dihydrokaempferol

Dihydroquercetin

Dihydromyricetin

(2R,3R)

Flavonol

Kaempferol

Quercetin

Myricetin

Oxidative transformation of flavan-3-ols

Transformation générique

(-)-catechin, (+)-epicatechin and (-)-epicatechin not accepted as substrates

requires (2R,3S) and at least two phenolic OH on ring B

Retention time (min)

with GSH 1mM

Retention time (min)

²

cyanidin

1. Ascorbate covalent adduct (m/z = 463.09)

2. Residual (+)-catechin (m/z = 291.08)

3. Dimer of oxidized catechin (m/z = 575.12)

Oxidative transformation of (+)-catechin

HPLC analysis of ANS final products derived from catechin

C18-reverse phase

Symmetrical dimer:

Structure already described

by Schofield’s group

with ANS from Arabidopsis

1. (+)-gallocatechin (no enzyme)

2’. Delphinidin (m/z = 575.12)

Oxidative transformation of (+)-gallocatechin

HPLC analysis of ANS final products derived from gallocatechin

C18-reverse phase

(+)-catechin : 100 µM

VvANS : 1 µM (Fe-loaded enzyme)

Ammonium acetate 20 mM pH 6.3

20 mM NaCl

1 mM 2-oxoglutarate

2 mM Ascorbate

T° = 22°C

Constant flux injection syringe

5 µL/min

capillary

Electrospray(positive

ionization)Extemporaneous

mixing

Kinetic monitoring of the reactional mediumPositive ionization – direct injection

Analyser (TOF)

Dead time ≈ 30 sec

total monitoring time≈ 50 min

MSMS/MS

Focus on catechin transformation

Production of the enzyme-Fe(II) complex

1) Incubation of holoenzyme in buffer with all cofactors + iron(II)

✓ Cofactors: 2-OG, Ascorbate, Iron salt (FeSO₄)

✓ Incubation time: 30 min, 35°C in Ammonium acetate (20 mM, pH 6,5)

✓ Final enzyme concentration: 10¯⁶ M (≈ 100 µg)

2) Removal of the iron in excess by gel filtration

❖ Gel-filtration cartridge: PD-10 (desalting column)

❖ Elution buffer: Ammonium acetate (20 mM, pH 6,5)

Containing 2-OG, ascorbate, but no iron salt

m/z 463.09

?

m/z 575.12

- 1 e-

- 2 e-

- 3 e-

- 4 e-

Effects of Glutathione GSHon VvANS transformation of (+)-catechin

Effects of Glutathione GSHon VvANS transformation of (+)-catechin

(GSH) = 0.5-10 mM

PKa (GSH/GS-) close to 9 E’0 GSH/GSSG = -0.23 V/ENH

HPLC detection of ANS final products derived from catechin

C18-reverse phase

with or without GSH

Retention time (min)

without GSH with GSH 1mM

Retention time (min)

²GSH-cyanidin covalent adduct

²

cyanidin

cyanidin

1. Ascorbate covalent adduct

2. Residual (+)-catechin

3. Dimer of oxidized catechin

➢ Disappearance of the ascorbate-cyanidin adduct➢ Disappearance of the symmetrical dimer➢ Production a GSH-cyanidin adduct➢ Marked increase in cyanidin➢ Much higher production yields

HPLC detection of ANS final products derived from catechinwith or without GSH

Catechin + VvANS: Real-time mass spectrometry

GSH adducts are most likely C4-thioethers

➢ The GSH adduct slowly decompose into cyanidin in acidic medium

➢ With gallocatechin, delphinidin is now replaced by a GSH adduct of

delphinidin

➢ VvANS may have been designed to produce GSH adducts of

anthocyanidins, possibly as stabilized precursors.

?

Overall conclusions

1. VvANS does not transform leucocyanidin isomers into cyanidin

They are only transformed into quercetin

2. The first generic step of the enzyme is C3-hydroxylation which

produces a 3,3-gem-diol intermediate

3. Dihydroflavonols of 2R,3R configuration are transformed into

Flavonols

Overall conclusions

1. VvANS does not transform leucocyanidin isomers into cyanidin

They are only transformed into quercetin

2. The first generic step of the enzyme is C3-hydroxylation which

produces a 3,3-gem-diol intermediate

3. Dihydroflavonols of 2R,3R configuration are transformed into

Flavonols

4. VvANS transforms gallocatechin into delphididin

5. VvANS gives very small amounts of cyanidin from catechin in the

absence of GSH.

In the presence of GSH 1 mM, It gives a GSH-cyanidin covalent

adduct in much higher yields which slowly decompose into

cyanidin at acidic pH

Gomez C, Conejero G, Torregrosa L, Cheynier V, Terrier N, Ageorges A.

In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the

contribution of anthoMATE transporters and GST.

Plant J. 2011 67(6):960-70.

➢ Is the production of GSH derived thioether of anthocyanidins

a physiological process ?

➢ Is it related to GST-mediated transport into vacuoles ?

Acknowledgments

Claudine TROSSAT

VvANS plasmid

construction and

transfection into E. coli

ISVV

Luc NEGRONI

Katell BATHANY

Mass Spectrometry

CBMN

Jia-Rong ZHANG

PhD Student

Polyphenol substrate Observed product

name m/z name m/z

Dihydroflavonol

(+)-Dihydrokaempferol (DHK) 289,05 Kaempferol 287.05

(+)-Dihydroquercetin (DHQ) 305,05 Quercetin 303.05

(+)-Dihydromyricetin (DHM) 321,05 Myricetin 319.05

Flavan-3-ol(s)

(+)-Catechin

without GSH

291,06

Dimer of

oxidized (+)-catechin575.11

Cyanidin 287.05

Adduct of

ascorbate with cyanidin463.07

(+)-Catechin

with GSH

291,06Adduct of

GSH with cyanidin594.13

Cyanidin 287.05

(+)-Gallocatechin

without GSH307,07 Delphinidin 303.04

(+)-Gallocatechin

with GSH307,07

Adduct of

GSH with delphinidin610.12

Delphinidin 303.04

Flavan-3,4-diol(s)

(+)-2,3-trans-3,4-cis-leucocyanidin

± GSH307,09 Quercetin 303.05

(+)-2,3-trans-3,4-trans-leucocyanidin

± GSH307,09

(+)-Dihydroquercetin 305.05

(-)-epidihydroquercetin 305.05

cyanidin 287.05

Quercetin 303.05

Activity of the iron-containing enzyme

with (+)-DHQ used as polyphenolic substrate

[Peak 3 (3’): (+)-DHK]

Active ANS can be reproducibly produced

ANS-Fe(II) complexNo iron salt

in the medium

ANS holoenzyme+ 10 µM iron saltin the medium

(+)-DHQ

(+)-epiDHQ

Quercetin

3. Production of 2,3-trans-3,4-cis-leucocyanidin

1) with dihydroflavonol reductase (DFR)

very weak yield

2) by reduction of (+)-Dihydroquercetin (DHQ)

with NaBH4 and acidic isomerisation (3,4 trans 3,4 cis)

much higher yield

✓ Purification by HPLC

• µBondapak C18 reverse phase : elution with 2% acetic acid

• Phenyl : elution with H2O

NMR Spectra of trans and cis-Leuco …