Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire.

The Journal of Biological Chemistry
Young-Wan KimS G Withers

Abstract

The Agrobacterium sp. beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-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 alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-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 th...Continue Reading

References

Oct 20, 1992·Biochemistry·J B Kempton, S G Withers
Jun 1, 1991·Journal of Bacteriology·U Kohlrausch, J V Höltje
Nov 1, 1995·Protein Science : a Publication of the Protein Society·C N PaceT Gray
Oct 1, 1995·Current Opinion in Structural Biology·G S Jacob
Jul 17, 1998·Current Opinion in Chemical Biology·D H Crout, G Vic
Jun 9, 1999·Current Opinion in Chemical Biology·J Minshull, W P Stemmer
Dec 11, 1999·Nucleic Acids Research·H M BermanP E Bourne
Mar 29, 2000·Accounts of Chemical Research·D L Zechel, S G Withers
Jun 21, 2001·Applied Microbiology and Biotechnology·K E JaegerM T Reetz
Mar 28, 1996·Chemical Reviews·Raymond A. Dwek
May 31, 2002·Bioscience, Biotechnology, and Biochemistry·Masayuki OkuyamaSeiya Chiba
Jun 11, 2002·The Journal of Organic Chemistry·Jakob F TolborgStephen G Withers
Jul 16, 2003·Biochemistry·Giuseppe PeruginoMarco Moracci
Nov 1, 2003·Bioorganic & Medicinal Chemistry Letters·Antonio TrinconeMarco Moracci
Dec 24, 2003·Trends in Biotechnology·Giuseppe PeruginoMarco Moracci
Jan 1, 1997·Methods in Enzymology·E A Merritt, D J Bacon

❮ Previous
Next ❯

Citations

Feb 20, 2007·Extremophiles : Life Under Extreme Conditions·Myoung-Hee LeeKwan-Hwa Park
Apr 29, 2005·Biomolecular Engineering·Ting WangTianhong Wang
May 11, 2011·Chemical Reviews·Gernot A StrohmeierMandana Gruber-Khadjawi
May 13, 2009·Journal of the American Chemical Society·Elise ChampionMagali Remaud-Siméon
Jun 16, 2009·Nature Chemical Biology·Susan M HancockStephen G Withers
Jul 25, 2006·Nature Methods·Amir AharoniStephen G Withers
Jun 3, 2006·Natural Product Reports·Andrew S Rowan, Chris J Hamilton
Oct 13, 2009·Journal of Biochemistry·Masafumi HidakaMotomitsu Kitaoka
Nov 11, 2008·Protein Engineering, Design & Selection : PEDS·Fankroma M T KonéCharles Tellier
Aug 16, 2012·Protein Engineering, Design & Selection : PEDS·Jae-Hoon ShimStephen G Withers
Apr 30, 2008·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Fathima Aidha Shaikh, Stephen G Withers
Aug 15, 2009·International Journal of Biological Sciences·Miranda MakiWensheng Qin
May 24, 2006·Bioscience, Biotechnology, and Biochemistry·Masahiro SugimuraMotomitsu Kitaoka
Nov 19, 2011·Biochemical and Biophysical Research Communications·Bo-Young YunNam-Chul Ha
Nov 18, 2008·Biochemical and Biophysical Research Communications·Ji-Hye ChoiKwan-Hwa Park
Feb 27, 2016·Journal of Agricultural and Food Chemistry·Hye-Yeon JeonJae-Hoon Shim
May 12, 2009·Journal of Biotechnology·Ronan M KellyHans Leemhuis
Jan 13, 2012·Protein Science : a Publication of the Protein Society·Salila PengthaisongJames R Ketudat Cairns
Mar 17, 2016·Journal of Agricultural and Food Chemistry·Hye-Won LeeJae-Hoon Shim
May 13, 2006·Biotechnology Advances·Y-H Percival ZhangJonathan R Mielenz
Apr 15, 2015·Journal of the American Chemical Society·David H KwanStephen G Withers
Oct 31, 2009·Chemistry & Biology·Beatrice Cobucci-PonzanoMarco Moracci
May 14, 2016·ACS Chemical Biology·Phillip M Danby, Stephen G Withers
Aug 15, 2006·Current Opinion in Chemical Biology·Susan M HancockStephen G Withers
Jun 24, 2006·Chembiochem : a European Journal of Chemical Biology·Johannes MülleggerStephen G Withers
Apr 19, 2008·Chemistry & Biology·David L Jakeman
Jul 25, 2017·Journal of the American Chemical Society·Phillip M Danby, Stephen G Withers
Sep 5, 2017·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Marc R Hayes, Jörg Pietruszka
Jul 13, 2005·Chembiochem : a European Journal of Chemical Biology·Antonio TrinconeMarco Moracci
Aug 8, 2018·Acta Crystallographica. Section F, Structural Biology Communications·Saioa UrrestiGideon J Davies
May 12, 2006·Organic & Biomolecular Chemistry·Young-Wan KimStephen G Withers
Sep 28, 2005·Organic & Biomolecular Chemistry·Steven J BroadwaterD Tyler McQuade
Sep 9, 2011·Natural Product Reports·Richard W GanttJon S Thorson
Apr 27, 2013·Journal of the Science of Food and Agriculture·Min-Su KimYoung-Wan Kim
Mar 11, 2011·Natural Product Reports·Christiane Luley-Goedl, Bernd Nidetzky
Feb 11, 2016·Biochemical Society Transactions·John B McArthur, Xi Chen
Jul 4, 2013·Biopolymers·Zachary Armstrong, Stephen G Withers

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.