Table S2. MALDI-TOF MS analysis of PrGT34B reaction products and hydrolysis of PrGT34B
galactosyltransferase reaction products by galactosidases.
(a) Relative abundance of ions corresponding to PrGT34B reaction products generated from linear
cellooligosaccharides
Donor substrate
Acceptor substrate
Relative abundance of ion species
[Glu]6
(m/z=1013)
[Glu]6[Xyl]1
(m/z=1145)
[Glu]6[Xyl]2
(m/z=1277)
[Glu]6[Xyl]3
(m/z=1409)
no donor substrate
1 mM UDP-xylose
4 mM UDP-xylose
cellohexaose
cellohexaose
cellohexaose
100.0%
60.4% ± 2.8%
2.2% ± 1.2%
[Glu]5
(m/z=851)
–
39.6% ± 2.8%
84.7% ± 5.0%
[Glu]5[Xyl]1
(m/z=983)
–
–
13.1% ± 3.8%
[Glu]5[Xyl]2
(m/z=1115)
–
–
–
[Glu]5[Xyl]3
(m/z=1247)
no donor substrate
1 mM UDP-xylose
4 mM UDP-xylose
cellopentaose
cellopentaose
cellopentaose
100.0%
63.0% ± 0.7%
15.3% ± 2.2%
[Glu]4
(m/z=689)
–
37.0% ± 0.7%
71.9% ± 3.7%
[Glu]4[Xyl]1
(m/z=821)
–
–
12.8% ± 1.5%
[Glu]4[Xyl]2
(m/z=953)
–
–
–
[Glu]4[Xyl]3
(m/z=1085)
no donor substrate
1 mM UDP-xylose
4 mM UDP-xylose
cellotetraose
cellotetraose
cellotetraose
100.0%
44.8% ± 4.5%
7.4% ± 1.7%
[Glu]6
(m/z=1013)
–
55.2% ± 4.5%
83.3% ± 2.6%
[Glu]6[Gal]1
(m/z=1175)
–
–
9.3% ± 1.4%
[Glu]6[Gal]2
(m/z=1337)
–
–
–
[Glu]6[Gal]3
(m/z=1499)
no donor substrate
1 mM UDP-galactose
4 mM UDP-galactose
cellotetraose
cellohexaose
cellohexaose
100.0%
57.0% ± 0.6%
22.8% ± 1.4%
–
43.0% ± 0.6%
73.0% ± 1.8%
–
–
4.3% ± 0.5%
–
–
–
(b) Relative abundance of ions corresponding to reaction products generated from pre-substituted
cellooligosaccharides
Assay condition
Donor substrate
Control (no protein)
PrGT34B
1 mM UDP-xylose
1 mM UDP-xylose
Assay condition
Donor substrate
Control (no protein)
PrGT34B
1 mM UDP-xylose
1 mM UDP-xylose
Pre-substituted cellohexaose as acceptor substrate
[Glu]6
(m/z=1013)
[Glu]6[Xyl]1
(m/z=1145)
[Glu]6[Xyl]2
(m/z=1277)
[Glu]6[Xyl]3
(m/z=1409)
2.7% ± 0.6%
2.2% ± 0.5%
85.4% ± 2.0%
57.4% ± 3.5%
12.0% ± 1.7%
38.9% ± 2.4%
–
1.4% ± 0.2%
Pre-substituted cellotetraose as acceptor substrate
[Glu]4
(m/z=689)
[Glu]4[Xyl]1
(m/z=821)
[Glu]4[Xyl]2
(m/z=953)
[Glu]4[Xyl]3
(m/z=1085)
7.0% ± 1.7%
2.6% ± 0.5%
83.1% ± 2.5%
71.5% ± 1.9%
9.9% ± 1.1%
25.9% ± 1.8%
–
–
(c) Relative abundance of ions corresponding to PrGT34B reaction products following hydrolysis with galactosidases
Hydrolyzing enzyme
Control (no enzyme)
β-D-Galactosidase
α-D-Galactosidase
Galactosylated cellohexaose as substrate
[Glu]6
(m/z=1013)
[Glu]6[Gal]1
(m/z=1175)
[Glu]6[Gal]2
(m/z=1337)
[Glu]6[Gal]3
(m/z=1499)
29.8% ± 1.9%
22.5% ± 3.1%
89.4% ± 2.0%
65.9% ± 1.6%
69.7% ± 3.1%
8.4% ± 1.5%
4.2% ± 1.1%
7.7% ± 1.3%
2.3% ± 0.6%
–
–
–
2
Footnotes: Reaction products were analyzed by MALDI-TOF MS. Masses of the monoisotopic
sodium adduct ions of the identified unsubstituted and glycosylated acceptor substrates are
indicated. Relative abundances were determined from signal intensities of all specific
monoisotopic sodium adduct ions for each spectrum. The relative abundances are shown as
percentages and are the arithmetic means (± SD) of values from at least three spectra for each
assay condition.
(a) Assays with IMAC purified protein extracts containing 20 µg protein PrGT34B, 1 mM or 4
mM UDP-sugar donor substrate and 1 mM cellohexaose, cellopentaose or cellotetraose acceptor
substrate were incubated for 17 h. Control reactions containing the acceptor substrate but no
protein and no donor substrate were treated accordingly.
(b) Assays with 8 µg protein PrGT34B or without protein as controls, 1 mM UDP-xylose and a
0.75 mM mixture of linear, mono- and dixylosylated cellooligosaccharide acceptor substrates
were incubated for 17 h.
(c) PrGT34B galactosyltransferase reaction products were used as substrates for glycoside
hydrolase reactions. Assays were incubated for 23 h with 0.1 U of either α-D-galactosidase or βD-galactosidase
or without enzyme (control).