Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM₃ demethylases

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 106; no. 39; pp. 16550 - 16555
Main Authors Lewis, Jared C, Bastian, Sabine, Bennett, Clay S, Fu, Yu, Mitsuda, Yuuichi, Chen, Mike M, Greenberg, William A, Wong, Chi-Huey, Arnold, Frances H
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 29.09.2009
National Acad Sciences
Subjects
Bacillus megaterium - enzymology
Bacillus megaterium - metabolism
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Biological Sciences
Carbohydrate Sequence
Catalysis
Cytochrome P-450 Enzyme System - chemistry
Cytochrome P-450 Enzyme System - metabolism
Kinetics
Molecular Sequence Data
Monosaccharides - chemistry
Monosaccharides - metabolism
Oxidoreductases, N-Demethylating - chemistry
Oxidoreductases, N-Demethylating - metabolism
Physical Sciences
Protein Conformation
Protein Engineering
Substrate Specificity
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Summary:Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450BM₃) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine.
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Author contributions: J.C.L., W.A.G., C.-H.W., and F.H.A. designed research; J.C.L., S.B., C.S.B., Y.M., and M.M.C. performed research; J.C.L., C.S.B., and Y.F. contributed new reagents/analytic tools; J.C.L., S.B., Y.M., and M.M.C. analyzed data; and J.C.L. and F.H.A. wrote the paper.
2Present address: Discovery Research Laboratories, Shionogi and Co. Ltd., 2–5-1 Mishima, Setu, Osaka 566-0022, Japan.
Contributed by Frances H. Arnold, August 10, 2009
1Present address: Department of Chemistry, Pearson Chemistry Laboratory, Tufts University, Medford MA, 02155.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0908954106