ndp sugar biosynthesis and glycorandomizationndp ‐ sugar biosynthesis and glycorandomization. by....
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Chemical Synthesis Community Lecture Series
NDP‐sugar biosynthesis and glycorandomization
ByAhmad H. Al‐Mestarihi
Graduate StudentLaboratory for Biosynthetic studies
04/26/2011
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Glycoslated Biomolecules and their functions
Types
Nucleic acids
Polysaccharides
Proteins
Lipids
Secondary metabolites
Functions
Information storage and transfer
Energy storage
Maintenance of cell structure
Molecular recognition
Virulence
Chemical defense
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significance of GlycoslatedBimolecules
some human diseases are associated with protein glycosylation patterns.
viral infections often involves recognition of specific cell surface protein glycoforms
bacterial virulence is related to cell‐surface polysaccharides
glycosylated small molecules are clinically useful for the treatment of bacterial and fungal infections, cancer, and other human diseases
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Glycoslated secondary metabolites
calicheamicin
rubradirin
everninimicin
kijanimicin
Cororubicinrespinomycin
Current Opinion in Chemical Biology 2008, 12:297–305
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Sugar Activation
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F‐6‐p
G‐6‐p
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The nucleotide moiety
Enzymatic recognitionGood leaving group for glycoslation
Thymidine diphosphate‐α‐D‐glucose:precursor for sugar modifications
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Entry point into TDP‐sugar metabolism
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
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Naturally occurring TDP‐sugarsGroup I
6‐deoxysugars
4‐amino‐4,6‐dideoxysugar 3‐amino‐3,6‐dideoxysugar
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Naturally occurring TDP‐sugarsGroup II
TDP‐D‐desosamine TDP‐D‐chalcomycin actinospectose
TDP‐4,6‐dideoxysugars
3‐amino‐2,3,6‐trideoxysugars
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Naturally occurring TDP‐sugarsGroup III
TDP‐2,6‐dideoxysugars
TDP‐2,3,6‐trideoxysugars
TDP‐4‐amino‐2,3,6‐trideoxysugars
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Other Naturally occurring nucleotide‐sugars
UDP‐sugars
GDP‐sugars
CDP‐sugars
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
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General Reaction Types of Sugar Biosynthetic Enzymes
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4,6‐dehydratase
Adrian D. Hegeman,‡ Jeffrey W. Gross,‡ and Perry A. Frey Biochemistry, Vol. 40, No. 22, 2001
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3,5‐epimerase
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TDP‐4‐keto‐6‐deoxy‐l‐mannose
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4‐aminotransferase
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external aldimine intermediate
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3‐C‐methyltransferase
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2‐dehydratase
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
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Unusual modification of NDP‐sugars
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
fortimycin, and moenomycin
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Unusual modification of NDP‐sugars
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
5‐methyl‐carboxypyrrole substituentmethyleurekanate moiety
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In vitro sugar biosynthesis(TDP‐L‐epivancosamine)
TDP‐L‐epivancosamine
Dehydration/deoxygenation Dehydration
transamination
methylationepimerization
4‐ketoreduction
Chen et al. PNAS October 24, 2000 vol. 97 no. 22 11947
Huawei Chen*, Michael G. Thomas, Brian K. Hubbard, Heather C. Losey, Christopher T. Walsh†, and Michael D. Burkart*Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115
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Production of TDP-L-epivancosamine via azideReduction
Hu Y, Al-Mestarihi A, Grimes CL, et al. J. Am Chem Soc. 2008, 130 (47): 15756-15757.
Extent of reduction > 95%
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0.00E+00
1.50E+07
3.00E+07
0 20 40 60 80 100
Cou
nts
Time (min)
Hu Y, Al-Mestarihi A, Grimes CL, et al. J. Am Chem Soc. 2008, 130 (47): 15756-15757.
OHO
OTDP
NO
Nitrososynthase Oxidation Reaction: Time Course
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Nitrososynthase Substrates
J. L. Vey, A. Al‐Mestarihi, Y. Hu, M. A. Funk, B. O. Bachmann and T. M. Iverson, Biochemistry, 2010, 49, 9306–9317.
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Glycosyltransferase structure
(a) The GT‐A fold is represented by the inverting enzyme SpsA from Bacillus subtilus, (b) the GT‐B fold, by bacteriophage T4 β‐glucosyltransferase
Lairson LL, H. B., Davies GJ, Withers SG. Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77, 521‐555 (2008).
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Glycosyltransferase
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Glycosyltransferase mechanism
Lairson LL, H. B., Davies GJ, Withers SG. Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77, 521‐555 (2008).
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Glycosyltransferase
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aryl‐C‐glycosidicbond formation
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Glycosyltransferase
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urdamycin
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Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
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in vitro and in vivo Glycorandomization
In vitro glycorandamization
Expensive cofactorsProtein purificationOptimazation of rxnconditions
In vivo glycorandamization
In vivo glycorandamization
Provided by host
Williams, GJ, Yang J, Zhang C, Thorson JS. ACS Chem. Biol. 2011, 6, 95‐100.
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TDP16 (glycosyltransferase) substrates
Williams, GJ, Yang J, Zhang C, Thorson JS. ACS Chem. Biol. 2011, 6, 95‐100.
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OleD active site residues
8 residues were chosen for mutation by saturation mutagenesis creating 8 mutant libraries
To maximize the efficiency of OleD for glycosylationwithin the cytoplasm of E. coli
Williams, GJ, Yang J, Zhang C, Thorson JS. ACS Chem. Biol. 2011, 6, 95‐100.
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Activity of prototype glycoside producing strains
Williams, GJ, Yang J, Zhang C, Thorson JS. ACS Chem. Biol. 2011, 6, 95‐100.
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Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
urdamycin
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Engineering of nucleotidyltransferase
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
E. coli galactokinase (GalK)
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Chemoenzymatic glycorandomization
GalK GalK
21 vancomycin analoguesN3‐289 39 additional vancomycin derivatives
d‐glucose andl‐vancosamine
Angew. Chem. Int. Ed. 2008, 47, 9814 – 9859
CuI‐catalyzed Huisgen[3+2]cycloaddition
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Summary
Structural diversity with a handful of enzyme activities
unique arrangements of catalytic residues, coenzymes, and cofactor
Deoxysugars can be further modified by unusual enzymes
Sugar modifying enzymes and glycosyltransferases exhibit a degree of substrate flexibility
Metabolic pathway engineering approaches resulted in new glycoforms
Characterizing and modifying the sugar biosynthetic enzymes will expand glycodiversification leading to generation of new compounds of interesting clinical use