Substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4 towards galactose, glucose, and their derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.03.095

Galactokinases (GalKs) have attracted significant research attention for their potential applications in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 presents a remarkably broad substrate range including 14 diverse natural and unnatural sugars. TLC and MS studies revealed that GalKSpe4 had relaxed activity towards galactose derivatives with modifications on the C-6, 4- or 2-positions. Additionally, GalKSpe4 can also tolerate glucose while glucose derivatives with modifications on the C-6, 4- or 2-positions were unacceptable. More interestingly, GalKSpe4 can phosphorylate l-mannose in moderate yield (43%), while other l-sugars such as l-Gal cannot be recognized by this enzyme. These results are very significant because there is rarely enzyme reported that can phosphorylate such uncommon substrates as l-mannose.

Full text of DOI:10.1016/j.bmcl.2012.03.095

Supplementary Materials
Substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4
towards galactose, glucose, and their derivatives
Yang Zoua,†, Wenjun Wangb,†, Li Caic, Leilei Chend, Mengyang Xuea, Xiaomei Zhanga,
Jie Shenb,*, Min Chena,*
a
The State Key laboratory of Microbial Technology, National Glycoengineering
Research Center and Department of Chemistry and Chemical Engineering, Shandong
University, Jinan, Shandong, 250100, China
b College of Pharmacy and the State Key Laboratory of Medicinal Chemical Biology,
Nankai University, Tianjin, 300071, China
c University of South Carolina Salkehatchie, Walterboro SC 29488, USA
d Institute of Agro-Food Science & Technology, Shandong Academy of Agricultural
Sciences, Jinan 250100, China
∗ Corresponding authors. Tel. / fax: +86‐531‐88366078.
E‐mail address: chenmin@sdu.edu.cn (M. Chen), jieshen@nankai.edu.cn (J. Shen).
† Contributed equally to this work.

Figure S1. TLC analysis of the GalKSpe4 reactions towards different sugars
Lane 1: GalKSpe4 reaction with compound 1 as substrate; lane 2: Reaction with
compound 1 as substrate without GalKSpe4; lane 3: GalKSpe4 reaction with
compound 2 as substrate; lane 4: Reaction with compound 2 as substrate without
GalKSpe4; lane 5: GalKSpe4 reaction with compound 3 as substrate; lane 6: Reaction
with compound 3 as substrate without GalKSpe4; lane 7: GalKSpe4 reaction with
compound 11 as substrate; lane 8: Reaction with compound 11 as substrate without
GalKSpe4; lane 9: GalKSpe4 reaction with compound 6 as substrate; lane 10:
Reaction with compound 6 as substrate without GalKSpe4; lane 11: GalKSpe4
reaction with compound 7 as substrate; lane 12: Reaction with compound 7 as
substrate without GalKSpe4; lane 13: GalKSpe4 reaction with compound 4 as
substrate; lane 14: Reaction with compound 4 as substrate without GalKSpe4; lane 15:
GalKSpe4 reaction with compound 5 as substrate; lane 16: Reaction with compound 5
as substrate without GalKSpe4; lane 17: ATP.

Figure S2. TLC analysis of the GalKSpe4 reactions towards different sugars.
Lane 1: GalKSpe4 reaction with compound 12 as substrate; lane 2: Reaction with
compound 12 as substrate without GalKSpe4; lane 3: GalKSpe4 reaction with
compound 13 as substrate; lane 4: Reaction with compound 13 as substrate without
GalKSpe4; lane 5: GalKSpe4 reaction with compound 14 as substrate; lane 6:
Reaction with compound 14 as substrate without GalKSpe4; lane 7: GalKSpe4
reaction with compound 15 as substrate; lane 8: Reaction with compound 15 as
substrate without GalKSpe4; lane 9: GalKSpe4 reaction with compound 29 as
substrate; lane 10: Reaction with compound 29 as substrate without GalKSpe4; lane
11: GalKSpe4 reaction with compound 30 as substrate; lane 12: Reaction with
compound 30 as substrate without GalKSpe4; lane 13: GalKSpe4 reaction with
compound 31 as substrate; lane 14: Reaction with compound 31 as substrate without
GalKSpe4; lane 15: GalKSpe4 reaction with compound 32 as substrate; lane 16:
Reaction with compound 32 as substrate without GalKSpe4; lane 17: GalKSpe4
reaction with compound 33 as substrate; lane 18: Reaction with compound 33 as
substrate without GalKSpe4.

Figure S3. TLC analysis of the GalKSpe4 reactions towards different sugars.
Lane 1: Reaction with compound 1 as substrate without GalKSpe4; lane 2: GalKSpe4
reaction with compound 1 as substrate; lane 3: Reaction with compound 2 as substrate
without GalKSpe4; lane 4: GalKSpe4 reaction with compound 2 as substrate; lane 5:
Reaction with compound 11 as substrate without GalKSpe4; lane 6: GalKSpe4
reaction with compound 11 as substrate; lane 7: Reaction with compound 6 as
substrate without GalKSpe4; lane 8: GalKSpe4 reaction with compound 6 as substrate;
lane 9: Reaction with compound 12 as substrate without GalKSpe4; lane 10:
GalKSpe4 reaction with compound 12 as substrate; lane 11: Reaction with compound
25 as substrate without GalKSpe4; lane 12: GalKSpe4 reaction with compound 25 as
substrate; lane 13: Reaction with compound 26 as substrate without GalKSpe4; lane
14: GalKSpe4 reaction with compound 26 as substrate; lane 15: Reaction with
compound 9 as substrate without GalKSpe4; lane 16: GalKSpe4 reaction with
compound 9 as substrate; lane 17: Reaction with compound 10 as substrate without
GalKSpe4; lane 18: GalKSpe4 reaction with compound 10 as substrate.

Figure S4. TLC analysis of the GalKSpe4 reactions towards different sugars.
Lane 1: Reaction with compound 27 as substrate without GalKSpe4; lane 2:
GalKSpe4 reaction with compound 27 as substrate; lane 3: Reaction with compound
28 as substrate without GalKSpe4; lane 4: GalKSpe4 reaction with compound 28 as
substrate; lane 5: Reaction with compound 17 as substrate without GalKSpe4; lane 6:
GalKSpe4 reaction with compound 17 as substrate; lane 7: Reaction with compound
18 as substrate without GalKSpe4; lane 8: GalKSpe4 reaction with compound 18 as
substrate; lane 9: Reaction with compound 19 as substrate without GalKSpe4; lane 10:
GalKSpe4 reaction with compound 19 as substrate; lane 11: Reaction with compound
20 as substrate without GalKSpe4; lane 12: GalKSpe4 reaction with compound 20 as
substrate; lane 13: Reaction with compound 21 as substrate without GalKSpe4; lane
14: GalKSpe4 reaction with compound 21 as substrate; lane 15: Reaction with
compound 22 as substrate without GalKSpe4; lane 16: GalKSpe4 reaction with
compound 22 as substrate; lane 17: Reaction with compound 34 as substrate without
GalKSpe4; lane 18: GalKSpe4 reaction with compound 34 as substrate.

Figure S5. TLC analysis of the GalKSpe4 reactions towards compound 23.
Lane 1: Reaction with compound 23 as substrate without GalKSpe4; lane 2:
GalKSpe4 reaction with compound 23 as substrate.

Figure S6. Mass spectrometry (-Q-ESI) of compound 2.

Figure S7. Mass spectrometry (-Q-ESI) of compound 3.

Figure S8. Mass spectrometry (-Q-ESI) of compound 4.

Figure S9. Mass spectrometry (-Q-ESI) of compound 5.

Figure S10. Mass spectrometry (-Q-ESI) of compound 6.

Figure S11. Mass spectrometry (-Q-ESI) of compound 7.

Figure S12. Mass spectrometry (-Q-ESI) of compound 12.

Figure S13. Mass spectrometry (-Q-ESI) of compound 13.

Figure S14. Mass spectrometry (-Q-ESI) of compound 14.

Figure S15. Mass spectrometry (-Q-ESI) of compound 15.

Figure S16. Mass spectrometry (-Q-ESI) of compound 31.

Dinitrosalicylic (DNS) acid assay
During the reaction process, 50 µL of reaction solution was taken out periodically
and transferred to 100 µL of DNS reagent solution (45 mM) to assess the reaction
progress. Each DNS reaction solution was placed in a bath of boiling water for 15 min
and subsequently cooled to room temperature followed by the addition of 15 µL of a
40% potassium sodiumtartrate solution to stabilize the color. The absorbance at 575
nm was recorded.
Scheme S1. Schematic reaction of DNS acid reagent and a reducing sugar

Figure S17. 400 MHz 1H NMR spectrum of L-Mannose-1-P in D2O.
[M‐H]‐
L‐Mannose‐1‐P
Figure S18. High resolution mass spectrometer (-Q-ESI) of L-Mannose-1-P.