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Structure of the ?-1,6/?-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121.

ABSTRACT: The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched ?-glucans with both ?-1,6- and ?-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-?N, a 118?kDa fragment of GTFA comprising residues 745-1763 and including the catalytic domain, was determined at 3.6?Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-?N has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-?N active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an ?-1,6/?-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128-1136 in GTFA-?N) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.

SUBMITTER: Pijning T

PROVIDER: S-EPMC3509963 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Structure of the α-1,6/α-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121.

Pijning Tjaard T Vujičić-Žagar Andreja A Kralj Slavko S Dijkhuizen Lubbert L Dijkstra Bauke W BW

Acta crystallographica. Section F, Structural biology and crystallization communications 20121114 Pt 12

The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched α-glucans with both α-1,6- and α-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-ΔN, a 118 kDa fragment of GTFA comprising residues 745-1763 and including the catalytic domain, was determined at 3.6 Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-ΔN has the s ...[more]

PMID: 23192022

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Project description:The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-?-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme.Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC.Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended ?-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Dataset Information

Structure of the ?-1,6/?-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121.

Publications

Structure of the α-1,6/α-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121.

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