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Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals alpha-Transglucosylase Activity in Glycoside Hydrolase Family 31
KTH, School of Biotechnology (BIO), Glycoscience.
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2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 52, 43288-43299 p.Article in journal (Refereed) Published
Abstract [en]

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these alpha-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in alpha-glucan side chain (de) branching, regulation of oligo-and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-alpha-glucan 4-alpha-glucosyltransferase from GH31. Distinct from 1,4-alpha-glucan 6-alpha-glucosyltransferases (EC and 4-alpha-glucanotransferases (EC, this enzyme strictly transferred one glucosyl residue from alpha(1 -> 4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-beta-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

Place, publisher, year, edition, pages
2012. Vol. 287, no 52, 43288-43299 p.
Keyword [en]
Thermotoga-Maritima Maltosyltransferase, Human Maltase-Glucoamylase, Escherichia-Coli, Crystal-Structure, Thermostable 4-Alpha-Glucanotransferase, Substrate-Specificity, Thermus-Aquaticus, Starch Metabolism, Cyclic Glucans, Action Pattern
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-116728DOI: 10.1074/jbc.M112.416511ISI: 000312940800010OAI: diva2:600623
Swedish Research CouncilFormas

QC 20130125

Available from: 2013-01-25 Created: 2013-01-25 Last updated: 2013-08-26Bibliographically approved
In thesis
1. Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Open this publication in new window or tab >>Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools.

In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes.

Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. viii, 82 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:13
CAZyme discovery, xyloglucan, polysaccharide-utilisation locus, microbiota, α-xylosidase, GH31, transglucosidase, human gut
National Category
Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-126956 (URN)978-91-7501-834-8 (ISBN)
Public defence
2013-09-13, Oskar Klein Auditoriet, FR4, 10:00, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 15:00 (English)

QC 20130826

Available from: 2013-08-26 Created: 2013-08-23 Last updated: 2016-01-26Bibliographically approved

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