P. Lafite, et al.
Molecular Catalysis 479 (2019) 110631
biosynthesis of S-glycoconjugates [25]. Along with ThuS and SunS,
involved in S-glycosylation of bacteriocin peptides [26–29], UGT74B1
from Arabidopsis thaliana is one of the only S-GTs involved in the nat-
ural biosynthesis of the most historically known S-glycosides, namely
glucosinolates [30,31]. Herein, we have studied UGT74B1 and identi-
fied the mechanism that leads to its specificity for S-glycosylation versus
O-glycosylation, using a diverse panel of acceptor substrates.
PATH, PPTH and hydroximates PAH, PPH were done using a tri-enzy-
matic assay that couples UDP formation with NADH consumption,
using pyruvate kinase and lactate dehydrogenase as enzymatic mixture.
Reactions conditions were identical to those reported previously [25].
Kinetics data were analyzed and fitted using Prism 4 (GraphPad).
2.4. Enzymatic assay (HPLC separation)
2
. Materials and methods
For thiophenols and phenols, an HPLC separation methodology was
used to quantify enzymatic activity. A reaction (200 μL total volume)
containing 1 mM UDP-glucose, acceptor (20 μL in 100% MeOH – final
concentrations ranging from 50 to 5000 μM) in 20 mM Tris buffer pH
8.0 was started by addition of 1 μM UGT74B1. The reaction was left for
2.1. Chemicals
All used chemicals and buffers were of the highest purity available.
HPLC solvents, phenols, thiophenols, pyruvate kinase, lactate dehy-
drogenase, phosphoenolpyruvate, NADH were purchased from Sigma-
Aldrich (Merck). Molecular biology and microbiology chemicals were
purchased from ThermoFisher. UDP-α-D-glucose was obtained from
Carbosynth Ltd (UK).
15 min at 37 °C, then 200 μL of quenching reagent (CH CN:HCOOH,
3
10:1) was added to the mixture. Proteins were precipitated by cen-
trifugation for 10 min at 10,000 rpm, and the supernatant was analyzed
by reverse-phase HPLC on
a Zorbax Eclipse XDB-C18 column
4.6 × 150 mm (Agilent) on an Agilent 1220 Infinity II LC System. The
mobile phase was delivered at a rate of 1 mL/min with a gradient from
1
13
H NMR and C NMR spectra of synthesized compounds were re-
corded on Bruker Avance II 400 or Bruker DPX 250 spectrometers.
Assignments are based on DEPT 135 sequence and on homo- and het-
eronuclear correlations. Chemical shifts are reported in parts per mil-
lion (ppm) from tetramethylsilane as the internal standard. Coupling
constants (J) are reported and expressed in Hertz (Hz); splitting pat-
terns are designated as br (broad), s (singlet), d (doublet), dt (doublet of
triplets), t (triplet) and m (multiplet). High-resolution mass spectra
A (0.1% HCOOH in H
2
O) to B (0.1% HCOOH in CH CN) (10% B for
3
4 min, 10% to 60% B in 10 min, 60% to 100% B in 2 min.). The column
effluent was monitored at 250 nm.
2.5. NMR structural study of desulfo-glucotropaeolin
NMR spectra of the glucosylated PATH product were recorded in
(
HRMS) were obtained with a Maxis Bruker 4 G instrument from the
DMSO-d solution at 313 K on a Bruker AVIII 500 spectrometer oper-
6
1 13
1 1 1 1 1 13
“
Fédération de Recherche” ICOA/CBM (FR2708) platform in the elec-
ating at 500.13 MHz for H and 125.13 MHz for C. 1D and 2D ex-
trospray ionization (ESI) mode. Infrared spectra of compounds were
recorded with a Thermo Scientific Nicolet iS10 spectroscope.
Preparation of PAH and PPH was adapted from published literature
periments (1D H, 2D 1H- H COSY, H- H NOESY, H- C HMQC and
HMBC) were run under TopSpin (version 3.2, Bruker Biospin,
1
Karlsruhe) with a BBFO { H, X} probe and a z gradient coil giving a
−1
1
13
[
32]. 1,1′-Carbonyldiimidazole (1.2 equiv.) was added to phenylpro-
maximum gradient of 50 G cm
.
H and C chemical shifts were re-
1
panoic acid or phenylacetic acid (6 mmol, 1 equiv.) dissolved in CH
20 mL) under argon atmosphere and the mixture was stirred at 22 °C
for 1 h. A solution of NH OH (50 wt. % in H O, 2 mL, 5 equiv.) was then
added and the reaction mixture was stirred at 22 °C for 20 h. After
3
CN
ferenced to the solvent residual signals of DMSO-d
6
(δ 2.49 for H and
(
39.70 ppm for 13C).
2
2
2.6. Saturation transfer difference
concentration under reduced pressure, H O (15 mL) was added to the
2
residue and the aqueous solution was extracted with EtOAc (3 x 15 mL).
The combined organic phases were washed with brine (1 x 25 mL),
STD spectra of the mixture of UGT74B1 (100μM) with UDPG
(1 mM) in phosphate buffer pH = 7.3 were undertaken on a Bruker
Avance III 600 equipped with CPTXI-cryoprobe. The experiments were
acquired with the standard Bruker stddiffesgp.3 sequence by using
trains of E-Burp-1 90° selective pulses. Selective pulses were applied at
0 ppm for the on resonance STD excitation, and -17.00 ppm for the
dried over Na
2
4
SO , filtered and evaporated under reduced pressure. The
residue was purified by Reveleris® column chromatography on C18
silica gel (H
2
O 100% to CH
3
CN 100%) to afford the desired products as
a slightly greenish powders.
1
PAH (Fig. S1): 682 mg, 75% yield. H NMR (250 MHz, DMSO-d
6
): δ
difference spectrum, with respect to H O at 4.70 ppm. The spectra were
2
1
2
0.59 (s, 1H, OH), 8.77 (s, 1H, NH), 7.28-7.14 (m, 5H, H-Ar), 3.26 (s,
measured with 8080 scans after eight dummy scans and lasted ap-
H, CH ). [33,34]
2
proximately 39 h.
1
PPH (Fig. S2): 761 mg, 65% yield). H NMR (400 MHz, DMSO-d ): δ
6
1
2
0.36 (s, 1H, OH), 8.70 (s, 1H, NH), 7.27-7.17 (m, 5H, H-Ar), 2.80 (t,
2.7. pKa determination by NMR
H, J =7.7 Hz, H-3), 2.25 (t, 2H, J =7.7 Hz, H-2). [33,34].
Thiohydroxamic acids, hydroxamic acids, and CTP were first char-
acterized by NMR on a Bruker Avance III 600 equipped with a CPTXI-
2.2. Cloning and expression of UGT74B1
1
cryoprobe. Then, H chemical shifts of these compounds were mon-
UGT74B1 was cloned as a histidine-tagged protein in E. coli and
itored for pH in order to determine their pKa in a 90:10 mixture of
purified as previously reported [25]. Site-directed mutageneses were
carried out using QuikChange II XL Site-Directed Mutagenesis kit
phosphate buffer pH 7.3 and methanol (CD
3
OH). The method consists
1
of making 1D H acquisitions following successive additions of acid or
base. Labile protons are not detectable under these experimental con-
ditions (phosphate buffer) because they undergo rapid exchange. The
chemical shift of the Hα protons closest to the deprotonation site is the
one which is followed by this method. A titration curve was established
by following the chemical shift of the chosen proton as a function of the
pH. The obtained data were fitted with an asymmetrical sigmoidal
function, which gave the value of the pKa.
(
Agilent) using WT-UGT74B1 plasmid as DNA template. Primers used
for site-directed mutagenesis are presented in Table S1 in Supplemen-
tary data. Mutagenesis products were directly transformed into XL10-
Gold ultracompetent cells (Agilent) by heat shock method. Each mutant
sequence DNA was sequenced (Eurofins genomics) and confirmed to be
identical to the known wild-type enzyme sequence DNA except the
targeted codon (either His22 and Asp113) replaced by alanine.
2.3. Enzymatic assay (Enzyme-coupled)
2.8. pKa calculations
Determination of enzymatic glycosylation for thiohydroximates
Phenols and thiophenols pKa were calculated using ACD/Labs
2