Directed evolution of a glycosynthase from agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire
Directed evolution of a glycosynthase from agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire
The Agrobacterium sp. ?-glucosidase (Abg) is a retaining ?-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ?-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ?-D-galactopyranosyl fluoride and 4-nitrophenyl ?-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.
42787-42793
Kim, Young-Wan
c5baeacc-14b4-452e-969e-d9f2132b5e32
Lee, Seung Seo
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb
Warren, R. Antony J.
a79742df-e469-4446-8a1b-12f60ce08d4f
Withers, Stephen G.
2fa507a8-6772-41a0-97a0-a33f443bbcc1
8 October 2004
Kim, Young-Wan
c5baeacc-14b4-452e-969e-d9f2132b5e32
Lee, Seung Seo
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb
Warren, R. Antony J.
a79742df-e469-4446-8a1b-12f60ce08d4f
Withers, Stephen G.
2fa507a8-6772-41a0-97a0-a33f443bbcc1
Kim, Young-Wan, Lee, Seung Seo, Warren, R. Antony J. and Withers, Stephen G.
(2004)
Directed evolution of a glycosynthase from agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire.
The Journal of Biological Chemistry, 279 (41), .
(doi:10.1074/jbc.M406890200).
Abstract
The Agrobacterium sp. ?-glucosidase (Abg) is a retaining ?-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ?-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ?-D-galactopyranosyl fluoride and 4-nitrophenyl ?-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.
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Published date: 8 October 2004
Organisations:
Organic Chemistry: SCF
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Local EPrints ID: 345656
URI: http://eprints.soton.ac.uk/id/eprint/345656
ISSN: 0021-9258
PURE UUID: 8a6040b9-77f3-41e7-be61-71c805d1038e
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Date deposited: 12 Feb 2013 16:43
Last modified: 15 Mar 2024 03:46
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Author:
Young-Wan Kim
Author:
R. Antony J. Warren
Author:
Stephen G. Withers
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