Supplementary Information
Substrates panel
The panel of substrates used for the screening was composed of glucose, galactose, fructose,
mannose, xylose, arabinose, N-acetyl-D-glucosamine, sucrose, maltose, lactose, sorbitol, xylitol,
cellobiose, cellotetraose, α-cyclodextrin, β-cyclodextrin, arabinan, chitosan, chitin, starch,
maltotetraose, glycerol, hydroxymethylfurfural.
Primers
D279E_fw-5'GAACGGATACAGCGTCGAGGGTGCTTATATCGGTG3’
D279E_rev-5'CACCGATATAAGCACCCTCGACGCTGTATCCGTTC3'
D279S_fw-5'-CTAAGAACGGATACAGCGTCAGTGGTGCTTATATCGGTGATG-3'
D279S_rev-5'-CATCACCGATATAAGCACCACTGACGCTGTATCCGTTCTTAG3´
D337E_fw-5'-CATACGACGCACATGAGAACTTCTACGCCAGCA-3'
D337E_rev-5'-TGCTGGCGTAGAAGTTCTCATGTGCGTCGTATG-3'
G270E_fw-5'-GAGATCACCATGCAGATGGAGGTCTCTAAGAACGGATACA-3'
G270E_rev-5'-TGTATCCGTTCTTAGAGACCTCCATCTGCATGGTGATCTC-3'
G321Y_fw-5'-CTTGTTACTCACTTCGCCTATGCTGGCGTCAACGTCAA-3'
G321Y_rev-5'-TTGACGTTGACGCCAGCATAGGCGAAGTGAGTAACAAG-3'
G321N_fw-5'-CTTGTTACTCACTTCGCCAATGCTGGCGTCAACGTCAA-3'
G321N_rev-5'-TTGACGTTGACGCCAGCATTGGCGAAGTGAGTAACAAG-3'
M170Y_fw-5'-CACGGAGGCTACGGATATGTCGCCCGCAAGCAC-3'
M170Y_rev-5'-GTGCTTGCGGGCGACATATCCGTAGCCTCCGTG-3'
S410R_fw-5'-CAATTCTACGACAGGGTTGCCGCCACTGC-3'
S410R_rev-5'-GCAGTGGCGGCAACCCTGTCGTAGAATTG-3'
F319S_fw-5'-GGTCTTGTTACTCACAGCGCCGGTGCTGGCG-3'
F319S_rev-5'-CGCCAGCACCGGCGCTGTGAGTAACAAGACC-3'
W373F_fw-5'-GCAGCAGCCACTCTTGGTTCTTGCAAATGGACATCACC-3'
W373F_rev-5'-GGTGATGTCCATTTGCAAGAACCAAGAGTGGCTGCTGC-3'
W373Y_fw-5'-GCAGCAGCCACTCTTGGTATTTGCAAATGGACATCACC-3'
W373Y_rev-5'-GGTGATGTCCATTTGCAAATACCAAGAGTGGCTGCTGC-3'
L403W_fw-5'-GTCCACCGTGACACCCTCTGGCTCTTCCAATTCTACGACA-3'
L403W_rev-5'-TGTCGTAGAATTGGAAGAGCCAGAGGGTGTCACGGTGGAC-3'
The double mutant ChitO Q268R/S410R was constructed using the wild-type pET SUMO/chito as
a template and both primers of Q268R and S410R. The previously constructed primer for Q268R
by (Heuts et al., 2007) was obtained and the primer sequences are Q268R_fw 5´CCTGCCGAGATCACCATGCGCATGGGTGTCTCTAAGAACG-3´ and Q268R_rev 5´CGTTCTTAGAGACACCCATGCGCATGGTGATCTCGGCAGG-3´. For the mutant
Q268R/G270E and Q268R/G270E/S410R an additional primer for Q268R/G270E was designed
since the two mutation sites Gln268 and Gly270 are too close to each other. The sequence of the
primers are Q268R/G270E_fw-5'-CTGCCGAGATCACCATGCGGATGGAG
GTCTCTAAGAACGGATAC-3' and Q268R/G270E_rev-5'-GTATCCGTTCTTAGAGACCTCC
ATCCGCATGGTGATCTC-3'. ChitO Q268R/G270E and the triple mutant Q268R/G270E/S410R
were constructed with wild-type pET SUMO-chito and pET SUMO-chito S410R as a template
respectively and the primers for Q268R/G270E were used.
Steady state kinetic plots for substrate-ChitO mutant combinations that display
substrate inhibition
Fig. S1. Steady-state kinetic data of ChitO mutants that displayed substrate inhibition.
Some mutants of ChitO showed different degrees of substrate inhibition as can be seen in the plots
above. Fitting of data was performed using the provided equation for substrate inhibition (See
Material and Methods).
Flavin absorbance spectra
absorbance
0.6
0.4
0.2
0.0
400
500
600
wavelength (nm)
Fig. S2. Comparison of flavin absorbance spectrum of wild-type ChitO (38.8 µM, taken from Heuts
et al. 2008) with the triple mutant ChitO Q268R/G270E/S410R (normalized for 38.8 µM).
The relatively high absorbance below 400 nm is due to the presence of the 4a-spirohydantoin FAD
degradation product formed during the purification process as has also been observed for sequencerelated the berberine bridge enzyme (Winkler et al., 2008). The content of this inactivated FAD
derivate in the ChitO preparations differed from batch to batch for wild-type and mutant enzymes.
However, enzyme concentrations were estimated based on the known extinction coefficient for the
oxidized FAD cofactor of wild-type ChitO at 445 nm, therefore only the concentration of intact
FAD, thus active enzyme, was used to determine the turnover rates.
References
-
-
Heuts DPHM, Janssen DB, Fraaije MW. 2007. Changing the substrate specificity of a
chitooligosaccharide oxidase from Fusarium graminearum by model-inspired site-directed
mutagenesis. FEBS Lett. 581:4905–9.
Winkler A, Lyskowski A, Riedl S, Puhl M, Kutchan TM, Macheroux P, Gruber K. 2008. A
concerted mechanism for berberine bridge enzyme. Nat. Chem. Biol., 12: 739–41.