SUPPORTING INFORMATION
Figure S1. Characteristics of the purified recombinant IRX10L.
(a) Purification of recombinant IRX10-L: lane 1, HEK293 cell culture medium; lane 2,
column flow through; lane 3, eluted protein; lane L, PageRuler Unstained Protein Ladder
(Thermo Scientific). The gel was stained with coomassie brilliant blue R-250 (Bio-Rad).
(b) RP-HPLC analysis of the products generated by incubating IRX10-L with UDP-Xyl
(2 mM), Xyl6-2AB (0.25 mM) and MgCl2 (1 mM) for 16 hours. The reaction products
(blue) and the products formed upon their treatment with endo-1,4-β-xylanase M1 (red)
were analyzed by RP-HPLC.
Quantification of the xylosyltranferase activity (c, d). The transfer of a Xyl residue from
UDP-Xyl results in the formation of UDP, which was quantified using a bioluminescent
UDP detection assay (UDP-GloTM, Promega).
(c) Xylosyltransferase activity with increasing amounts of purified IRX10L (0-1.25 g)
in the presence (dashed line) or absence (solid line) of exogenous Xyl6 acceptor (1 mM)
and UDP-Xyl (0.8 mM). Data include average ±SD (n=3).
(d) Acceptor substrate specificity of IRX10-L. Xylosyltransferase reactions consisted of
HEPES-HCl (75 mM), pH 6.8, containing IRX10-L (1.0 M), UDP-Xyl (65 M), MgCl2
(2.5 mM) and potential acceptor substrates, including xylooligomers ranging from
monomeric Xyl to Xyl6 and cellopentaose (Glc5) (100 M). Data are averages of
duplicate reactions.
IRX10-L does not catalyze xylan chain initiation or the transfer of xylosyl residues to
Glc5, as addition of IRX10-L did not result in the production of UDP, which would be
consistent with such catalytic activities.
Figure S2. Xylosyltransferase activity assays of several enzymes predicted to be
involved in xylan synthesis. The enzymes were heterologously expressed in human
embryonic kidney cells, purified and incubated with an activated sugar donor (UDP-Xyl;
2 mM), a chemically labeled acceptor (Xyl6-2AB; 0.25 mM) and MgCl2 (1mM). The
reaction products were analyzed by MALDI-TOF MS after 16 hours. IRX10-L
transferred several xylosyl residues from UDP-Xyl to the acceptor. Heterologously
expressed and purified IRX9 and IRX14 alone or in combination did not show xylosyl
transferase activity under these conditions or when MnCl2 (1 mM) was added to the
standard assay.
Figure S3. Structural characterization of the products of the reaction catalyzed by
IRX10-L. The enzyme was incubated with unlabeled β-(1→4)-xylohexaose (100 g) and
UDP-Xyl for 120 hours.
(a) MALDI-TOF MS analysis of the reaction products. Labels correspond to the
[M+Na]+ and [M+K]+ ions.
(b) Partial gradient-selected correlation spectroscopy (gCOSY) NMR spectrum of the
reaction products containing the spin system (labeled with blue dotted lines) of internal
(Int) β-(1→4) linked xylosyl residues. The other resonances in the spectrum correspond
to terminal (Ter) β-Xyl and reducing (Red) α-and β-Xyl (see labels in the 1D 1H
spectrum in the top).
These results show that the products of the reaction catalyzed by IRX10-L are b-(1→4)
xylooligosaccharides with DP 7 to 16.
Figure S4. Heteronuclear 1H-13C NMR characterization of the products generated by
IRX10-L. The enzyme was incubated with UDP-Xyl and unlabeled β-(1→4)xylohexaose (100 g) for 120 hours.
(a) 13C resonances in the gradient-selected heteronuclear single-quantum coherence
(gHSQC) spectrum were assigned by correlation to 1H resonances in the gCOSY
spectrum (Figure S3b). The chemical shifts of C1 and C4 of internal β-(1→4) linked
Xylp residues are indicated by dashed horizontal lines.
(b) The gradient-selected heteronuclear multiple-bond correlation (gHMBC) spectrum
contained strong cross-peaks (red boxes) due to three-bond interglycosidic scalar
couplings from C1 to H4’ (3JC1,H4’) and from H1 to C4’ (3JH1,C4’) of two xylosyl residues
connected by a β -(1→4) linkage.
Figure S5. Determination of the optimal pH for xylosyltransferase activity. IRX10-L
was incubated with UDP-Xyl (2 mM), Xyl6-2AB (0.25 mM) and MgCl2 (1 mM). The
reactions were performed in various buffers, including MES-KOH (50 mM) (pH 5.5-6.7)
and HEPES (50 mM) (pH 6.8-8.0). Analysis of the reaction products by MALDI-TOF
MS shows that the xylosyl transferase activity is optimal at pH 6.7-6.8.
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Figure S6. Substrate specificity of the purified recombinant TBL29/ESK1.
(a) Purification of recombinant TBL29/ESK1: Lane 1, HEK293 cell culture medium; lane
2, column flow through; lane 3, eluted protein; lane L, Benchmark Protein ladder (Life
Technologies). The gel was stained with coomassie brilliant blue R-250 (Bio-Rad).
MALDI-TOF MS analysis of the products after incubating TBL29/ESK1 for 16 hours
with acetyl-CoA as a donor and a chemically labeled acceptor, (b) Xyl6-2AB and (c)
Man5-2AB.
Figure S7. Characterization by NMR of the products of the reaction catalyzed by
TBL29/ESK1.
(a) gCOSY spectrum of the acceptor substrate β-(1→4)-xylohexaose (Xyl6, 100 g)
before incubation with the enzyme.
(b) Analysis of the products generated by incubating β-(1→4)-xylohexaose with
TBL29/ESK1 and acetyl-CoA for 48 hours. The gCOSY spectrum of the reaction
products contained the characteristic resonances of xylosyl residues bearing an acetyl
group at O-2 (X2Ac; blue) and at O-3 (X3Ac; red). Two spin systems were observed for
xylosyl residues acetylated at O-2 (X2Ac) and two other spin systems were observed for
xylosyl residues acetylated at O-3 (X3Ac). The spin systems in each pair were assigned
to internal and terminal xylosyl residues acetylated at the same position. Multiple H1:H2
cross-peaks were also observed for non-acetylated terminal and internal xylosyl residues
because their chemical shifts are affected by the presence of O-acetyl substituents on
nearby residues. These results show that TBL29/ESK1 catalyzes the transfer of O-acetyl
groups from acetyl-CoA to O-2 or O-3 of xylosyl residues of β-(1→4)
xylooligosaccharides.
Figure S8. Determination of the regiospecificity of TBL29/ESK1. The enzyme was
incubated with xylohexaose and acetyl-CoA for 12 hours and the course of the reaction
was monitored by real-time 1H NMR. The resonance (δ 2.168) corresponding to the
methyl protons of the acetyl groups attached to O-2 (X2Ac) appeared early during the
course of the reaction. As the reaction progressed, the resonance (δ 2.154) of acetyl
groups attached to O-3 (X3Ac) increased and became dominant. (See also Figure 3c) The
integral of the resonance at δ1H 2.175 (XAc) increased in parallel to the X3Ac resonance,
lagging behind the X2Ac integral, indicating that it corresponds to an acetyl-migration
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product. Based on this observation and its low abundance, it was tentatively assigned as
the methyl proton of acetyl group on O-4 of the terminal Xyl residue.
Figure S9. Effect of divalent metals on the acetyltransferase activity of TBL29/ESK1. To
determine metal dependence, the enzyme was dialyzed against buffer containing Chelex100 to remove any contaminating divalent cations prior to activity assays. The reactions
were performed with no additives or with EDTA added to a final concentration of 5 mM.
Reactions were equilibrated for 30 min, then initiated by the addition of Xyl6-2AB
acceptor substrate. Acetyltransferase reactions consisted of HEPES-HCl (75 mM), pH
6.8, containing TBL29/ESK1 (4 µM), acetyl-CoA (1 mM), Xyl6-2AB acceptor substrate
(0.25 mM) with or without EDTA (5 mM). Analysis of the reaction products by MALDITOF MS indicated that ESK1/TBL29 does not require any cofactors for activity.
Figure S10. Effect of acceptor substrate O-acetylation on the xylosyltransferase activity
of IRX10-L. MALDI-TOF MS was used to analyze the reaction products formed by
incubating Xyl6-2AB (0.25 mM), UDP-Xyl (2mM) and acetyl-CoA (2 mM) with:
(a) IRX10-L and TBL29/ESK1 together for 40 hours.
(b) IRX10-L for 16 hours after which TBL29/ESK1 was added and incubated for an
additional 24 hours.
(c) TBL29/ESK1 for 16 hours after which IRX10-L was added and incubated for an
additional 24 hours. The [M+H]+ ion series at m/z 1063, 1195 and 1328 correspond to
non-acetylated oligosaccharides generated by IRX10-L.
Addition of each acetyl group by TBL29/ESK1 increases the mass of the oligosaccharide
by 42 Da. The number of O-acetyl substituents is indicated by asterisks. These results
indicate that the presence of O-acetyl substituents limits the IRX10-L catalyzed extension
of the xylooligosaccharide acceptor.
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