Affiliation: Department of Chemistry
Email Address: withers[at]chem.ubc.ca
Phone: 604 822-3402
Coming from a background in the investigation of enzyme mechanisms, protein structure/function relationships, enzyme inhibitor design/synthesis and protein engineering we are increasingly adopting and developing tools to carry out such studies in a high-throughput manner. Our particular interest lies in enzymes that synthesise and degrade glycoconjugates such as glycolipids and glycoproteins and polysaccharides such as cellulose and starch.
One major area of interest is in the use of directed evolution approaches to engineer enzymes for the efficient synthesis of glycoconjugates, both by evolving natural glycosyl transferases and through the engineering of Glycosynthases, which are mutant glycosidases developed by our laboratory. To that end we are developing high-throughput screens that allow more than a million transformed bacteria to be scanned per hour. Enzymes so developed have now been licensed by industry for commercial syntheses.
Another area of interest is in the discovery of enzyme inhibitors or chaperones as novel pharmaceuticals through high-throughput screening. Recent successes include potential treatments for diabetes and influenza. In each case we are targeting key carbohydrate synthesizing or degrading enzymes.
A third area of interest is in the development of new, activity-based proteomics reagents to probe the presence and identities of specific families of enzymes within whole proteomes. We have recently published the first quantitative measures of this type using ICAT-like reagents. Given our deep experience with the enzymes involved in cellulose degradation we are applying these probes to better understanding of cellulosic biomass conversion for biofuel purposes and are partnering with a leading biofuels company in this process.
A new direction for our laboratory involves a collaboration with Prof. Steven Hallam on enzyme discovery through metagenomic approaches. Our focus has been on the development of new, much higher-throughput activity-based screens. This should allow us to find many more enzymes of a wider range of activities.
Goddard-Borger, E. D., Tropak, M. B., Yonekawa, S., Tysoe, C. R., Mahuran, D. J. and Withers, S. G. “Rapid Assembly of a Library of Lipophilic Iminosugars via the Thiol-ene Reaction Yields Promising Pharmacological Chaperones for the Treatment of Gaucher Disease” (2012) J Med Chem 55, 2737-2745. Pubmed
Rempel, B. P., Tropak, M. B., Mahuran, D. J., Withers, S. G “Tailoring the Specificity and Reactivity of a Mechanism-Based Inactivator of Glucocerebrosidase for Potential Therapeutic Applications” (2011)Angew Chemie, 50, 10381-10383. Pubmed
Kwan, D. H., Chen, H., Ratananikom, K., Hancock, S. M., Watanabe, Y., Kongsaeree, P. T., Samuels, A. L. and Withers, S. G. “Self-immobilizing fluorogenic imaging agents of enzyme activity” (2011)Angewandte Chemie, 50, 300-303. Pubmed
Phenix, C. P., Rempel, B. P., Colobong, K., Doudet, D. J., Adam, M. J., Clarke, L. A. and Withers, S. G. (2010) “Imaging enzyme replacement therapy for Gaucher Disease with PET” Proceedings of the National Academy of Sciences of the Unites States of America 107, 10842-7. PubMed
Rich, J. R. and Withers, S. G. (2009) “Emerging methods for the production of homogeneous human glycoproteins” Nature Chemical Biology 5, 206-15. PubMed
Rao, F. V., Rich, J. R., Rakic, B., Buddai, S., Schwartz, M., Johnson, K., Bowe, C., DeFrees, S, Wakarchuk, W. W., Withers, S. G., Strynadka, N. C. J. (2009) “Structural insight into mammalian sialyltransferases”Nature Structural and Molecular Biology 16, 1186-8. PubMed
Engineering and Evolution
Shim, J-H., Chen, H., Rich, J. R., Goddard-Borger, E. D. and Withers, S. G. “Directed evolution of a β-glucosidase from Agrobacterium sp. enhances its glycosynthase catalytic activity toward C3-OH modified donor sugar” (2012) PEDS, 25, 465-472. Pubmed
Rich, J. R. and Withers, S. G. “A Chemoenzymatic Total Synthesis of the Neurogenic Starfish Ganglioside LLG-3 Using an Engineered and Evolved Synthase” (2012) Angewandte Chemie, 51, 1-5. Pubmed
Yang, G., Rich, J. R., Gilbert, M., Wakarchuk, W. W., Feng, Y., Withers, S. G. (2010) “Fluorescence activated cell sorting as a general ultra high-throughput screening method for directed evolution of glycosyltransferases” Journal of the American Chemical Society 132, 10570-7. Pubmed
Reitinger, S., Yu, Y., Wicki, J., Ludwiczek, M., D’Angelo, I., Baturin, S., Okon, M., Strynadka, N.C.J., Lutz, S., Withers, S. G., McIntosh, L. P. (2010) “Circular permutation of Bacillus circulans< Xylanase: A kinetic and structural study” Biochemistry 49, 2464-74. PubMed
Hancock, S. M., Rich J. R., Caines, M. E. C., Strynadka, N. C. J. and Withers, S. G. (2009) “Designer enzymes for glycosphingolipid synthesis by directed evolution” Nature Chemical Biology 5, 508-14. PubMed
Yang, G and Withers, S. G. (2009) “Ultrahigh-throughput FACS-based screening for directed enzyme evolution” ChemBioChem 10, 2704–15. PubMed
Lairson, L. L., Watts, A. G., Wakarchuk, W. W. & Withers, S. G. (2006) “Glycosyl transferase substrate engineering: harnessing substrate promiscuity and expanding biological catalysis through transient chemical modification” Nature Chemical Biology 2, 724-8.
Aharoni, A., Thieme, K., Chiu, C. P.C., Buchini, S., Lairson, L. L., Chen, H., Strynadka, N.C.J., Wakarchuk, W.W. and Withers, S.G. (2006) “High-throughput screening methodology for the directed evolution of glycosyltransferases.” Nature Methods 3, 609-14. PubMed
Vaughan, M. D., Johnson, K., DeFrees, S., Tang, X., Warren, R. A. J., Withers, S. G. (2006) “Glycosynthase-mediated synthesis of glycosphingolipids” Journal of the American Chemical Society 128, 6300-01.PubMed
High-throughput screening for inhibitors
Kuntz, D. A., Tarling, C. A, Stick, R. V., Withers, S. G. and Rose, D. R. (2008) “Structural analysis of novel Golgi α-mannosidase II inhibitors from a focused glycosidase inhibitor screen.” Biochemistry 47, 10058-68. PubMed
Tarling, C. A., Woods, K., Brastianos, H. C., Zhang, R., Brayer, G. D., Andersen, R. J. and Withers, S. G. (2008) “The search for novel human pancreatic α-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts” ChemBioChem. 9, 433-8. PubMed
Tropak, M.B., Blanchard, J. E., Brown, E. D. and Mahuran, D. (2007) “High throughput screening for human lysosomal β-N-acetyl hexosaminidase inhibitors acting as pharmacological chaperones” Chemistry & Biology 14, 153-64. PubMed
Hekmat, O., He, S., Warren, R. A. J. and Withers, S. G. (2008) “A mechanism-based ICAT strategy for comparing relative expression and activity levels of glycosidases in biological systems” Journal of Proteome Research 7, 3282-92. PubMed
Hekmat, O., Florizone, C., Kim, Y-W., Eltis, L. D., Warren, R. A. J. and Withers, S. G. (2007) “Specificity fingerprinting of retaining α-1,4-glycanases in Cellulomonas fimi secretome using two fluorescent mechanism-based probes” ChemBioChem 8, 2125-32. PubMed
Williams, S. J., Hekmat, O. and Withers, S. G. (2006) “Synthesis and testing of mechanism-based protein profiling probes for retaining endo-glycosidases” ChemBioChem 7, 116-24. PubMed
Hekmat, O, Kim, Y-W, Williams, S. J., He, S. and Withers, S. G. (2005) “Active-site peptide ‘fingerprinting’ of glycosidases in complex mixtures by mass spectrometry: Discovery of a novel retaining β-1,4-glycanase in Cellulomonas fimi” Journal of Biological Chemistry 280, 35126-35. PubMed
History:Member for 4 years 16 weeks