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Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
KTH, School of Biotechnology (BIO), Glycoscience.
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:13
Keyword [en]
CAZyme discovery, xyloglucan, polysaccharide-utilisation locus, microbiota, α-xylosidase, GH31, transglucosidase, human gut
National Category
Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-126956ISBN: 978-91-7501-834-8 (print)OAI: oai:DiVA.org:kth-126956DiVA: diva2:642872
Public defence
2013-09-13, Oskar Klein Auditoriet, FR4, 10:00, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 15:00 (English)
Opponent
Supervisors
Note

QC 20130826

Available from: 2013-08-26 Created: 2013-08-23 Last updated: 2016-01-26Bibliographically approved
List of papers
1. Structural and enzymatic characterization of a glycoside hydrolase family 31 alpha-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification
Open this publication in new window or tab >>Structural and enzymatic characterization of a glycoside hydrolase family 31 alpha-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification
Show others...
2011 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 436, 567-580 p.Article in journal (Refereed) Published
Abstract [en]

The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, alpha-xylosidases, beta-galactosidases and alpha-L-fucosidases, among others. In the present paper, we show the characterization of Xy131A, a key alpha-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXy131A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-beta-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXy131A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicas, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.

Keyword
enzymology, hemicellulose, plant cell wall, saccharification, xyloglucan
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-37169 (URN)10.1042/BJ20110299 (DOI)000292488500006 ()21426303 (PubMedID)2-s2.0-79957714498 (Scopus ID)
Note

QC 20110802

Available from: 2011-08-02 Created: 2011-08-02 Last updated: 2017-12-08Bibliographically approved
2. The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases
Open this publication in new window or tab >>The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17Article in journal (Refereed) Published
Abstract [en]

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed beta-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-179859 (URN)10.1074/jbc.M110.215962 (DOI)000289788300076 ()2-s2.0-79955415550 (Scopus ID)
Note

QC 20160126

Available from: 2016-01-04 Created: 2016-01-04 Last updated: 2017-12-01Bibliographically approved
3. NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 a-Xylosidase
Open this publication in new window or tab >>NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 a-Xylosidase
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2012 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 18, no 42, 13395-13404 p.Article in journal (Refereed) Published
Abstract [en]

The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass-resource utilization. Recent molecular modeling of the a-xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme-kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F. M. Ibatullin, A. Nakhai, H. J. Gilbert, G. J. Davies, H. Brumer, Biochem. J. 2011, 436, 567580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR-NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active-site variants and xyloglucan hexa- and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello-tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant a-(1?6)-linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.

Keyword
a-xylosidases, Cellvibrio japonicus, molecular recognition, mutagenesis, NMR spectroscopy, xyloglucan
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-105659 (URN)10.1002/chem.201200488 (DOI)000309597000023 ()
Funder
Swedish Research Council
Note

QC 20121129

Available from: 2012-11-29 Created: 2012-11-23 Last updated: 2017-12-07Bibliographically approved
4. Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals alpha-Transglucosylase Activity in Glycoside Hydrolase Family 31
Open this publication in new window or tab >>Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals alpha-Transglucosylase Activity in Glycoside Hydrolase Family 31
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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 2.4.1.24) and 4-alpha-glucanotransferases (EC 2.4.1.25), 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.

Keyword
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
Identifiers
urn:nbn:se:kth:diva-116728 (URN)10.1074/jbc.M112.416511 (DOI)000312940800010 ()
Funder
Swedish Research CouncilFormas
Note

QC 20130125

Available from: 2013-01-25 Created: 2013-01-25 Last updated: 2017-12-06Bibliographically approved
5. A discrete genetic locus confers select Bacteriodetes with a niche role in xyloglucan metabolism in the human gut
Open this publication in new window or tab >>A discrete genetic locus confers select Bacteriodetes with a niche role in xyloglucan metabolism in the human gut
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-126962 (URN)
Note

QS 2013

Available from: 2013-08-23 Created: 2013-08-23 Last updated: 2013-08-26Bibliographically approved
6. Identification and characterization of a locus in Cellvibrio japonicus involved in xylogluco-oligosaccharide saccharification
Open this publication in new window or tab >>Identification and characterization of a locus in Cellvibrio japonicus involved in xylogluco-oligosaccharide saccharification
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-126960 (URN)
Note

QS 2013

Available from: 2013-08-23 Created: 2013-08-23 Last updated: 2013-08-26Bibliographically approved
7. Screening and activity profiling of the glycoside hydrolase family 31 members from Bacteroides thetaiotaomicron reveal novel specificities
Open this publication in new window or tab >>Screening and activity profiling of the glycoside hydrolase family 31 members from Bacteroides thetaiotaomicron reveal novel specificities
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-126961 (URN)
Note

QS 2013

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

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