ndp sugar biosynthesis and glycorandomizationchemical synthesis community lecture series ndp ‐...
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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
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
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
Glycoslated secondary metabolites
calicheamicin
rubradirin
everninimicin
kijanimicin
Cororubicinrespinomycin
Current Opinion in Chemical Biology 2008, 12:297–305
Sugar Activation
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F‐6‐p
G‐6‐p
The nucleotide moiety
Enzymatic recognitionGood leaving group for glycoslation
Thymidine diphosphate‐α‐D‐glucose:precursor for sugar modifications
Entry point into TDP‐sugar metabolism
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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
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General Reaction Types of Sugar Biosynthetic Enzymes
4,6‐dehydratase
Adrian D. Hegeman,‡ Jeffrey W. Gross,‡ and Perry A. Frey Biochemistry, Vol. 40, No. 22, 2001
3,5‐epimerase
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TDP‐4‐keto‐6‐deoxy‐l‐mannose
4‐aminotransferase
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external aldimine intermediate
3‐C‐methyltransferase
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2‐dehydratase
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Unusual modification of NDP‐sugars
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fortimycin, and moenomycin
Unusual modification of NDP‐sugars
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5‐methyl‐carboxypyrrole substituentmethyleurekanate moiety
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
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%
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
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.
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).
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).
Glycosyltransferase
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aryl‐C‐glycosidicbond formation
Glycosyltransferase
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urdamycin
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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.
TDP16 (glycosyltransferase) substrates
Williams, GJ, Yang J, Zhang C, Thorson JS. ACS Chem. Biol. 2011, 6, 95‐100.
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.
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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urdamycin
Engineering of nucleotidyltransferase
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E. coli galactokinase (GalK)
Chemoenzymatic glycorandomization
GalK GalK
21 vancomycin analoguesN3‐289 39 additional vancomycin derivatives
d‐glucose andl‐vancosamine
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CuI‐catalyzed Huisgen[3+2]cycloaddition
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