Optimizing Glycosyltransferase Specificity via "Hot Spot" Saturation Mutagenesis Presents a Catalyst for Novobiocin Glycorandomization

Gavin J. Williams; Randal D. Goff; Changsheng Zhang; Jon S. Thorson

doi:10.1016/j.chembiol.2008.02.017

Optimizing Glycosyltransferase Specificity via "Hot Spot" Saturation Mutagenesis Presents a Catalyst for Novobiocin Glycorandomization

Gavin J. Williams, Randal D. Goff, Changsheng Zhang, Jon S. Thorson

Research output: Contribution to journal › Article › peer-review

87 Scopus citations

Abstract

A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

Original language	English
Pages (from-to)	393-401
Number of pages	9
Journal	Chemistry and Biology
Volume	15
Issue number	4
DOIs	https://doi.org/10.1016/j.chembiol.2008.02.017
State	Published - Apr 21 2008

Bibliographical note

Funding Information:
We are grateful to the School of Pharmacy Analytical Instrumentation Center for analytical support. This work was supported in part by National Institutes of Health grants AI52218 and U19 CA113297. J.S.T is a University of Wisconsin H.I. Romnes Fellow. The authors report competing interests. J.S.T. is a cofounder of Centrose (Madison, WI).

Funding

We are grateful to the School of Pharmacy Analytical Instrumentation Center for analytical support. This work was supported in part by National Institutes of Health grants AI52218 and U19 CA113297. J.S.T is a University of Wisconsin H.I. Romnes Fellow. The authors report competing interests. J.S.T. is a cofounder of Centrose (Madison, WI).

Funders	Funder number
National Institutes of Health (NIH)	U19 CA113297
National Institute of Allergy and Infectious Diseases	R01AI052218

Keywords

CHEMBIO
PROTEINS

ASJC Scopus subject areas

Biochemistry
Molecular Medicine
Molecular Biology
Pharmacology
Drug Discovery
Clinical Biochemistry

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10.1016/j.chembiol.2008.02.017

Cite this

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abstract = "A comprehensive two-phase {"}hot spot{"} saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.",

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AU - Goff, Randal D.

AU - Zhang, Changsheng

AU - Thorson, Jon S.

N1 - Funding Information: We are grateful to the School of Pharmacy Analytical Instrumentation Center for analytical support. This work was supported in part by National Institutes of Health grants AI52218 and U19 CA113297. J.S.T is a University of Wisconsin H.I. Romnes Fellow. The authors report competing interests. J.S.T. is a cofounder of Centrose (Madison, WI).

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N2 - A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

AB - A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

KW - CHEMBIO

KW - PROTEINS

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JO - Chemistry and Biology

JF - Chemistry and Biology

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ER -

Optimizing Glycosyltransferase Specificity via "Hot Spot" Saturation Mutagenesis Presents a Catalyst for Novobiocin Glycorandomization

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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