Crystal Structure and Site-directed Mutagenesis of Bacillus macerans Endo-1,3
–1,4-
β-glucanase

Hahn, Michael G.; Olsen, Ole; Politz, Oliver; Borriss, Rainer; Heinemann, Udo

doi:10.1074/jbc.270.7.3081

Cited by 113 publications

(97 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…These domains bind to substrate on their own and recruit it to the enzymatically active domain (45). In the case of cellulase 9B (cellulose 1,4-β-endoglucanase) from Cellulomonas, both carbohydratebinding domains have a similar fold as bacterial 1,3-1,4-β-glucanases, but lack catalytic residues (46)(47)(48), reminiscent of the situation with GtfB. We propose that catalytically inactive subunits/domains may have evolved from active enzymes to facilitate substrate recruitment of carbohydrate-modifying enzymes.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of a cytosolic O -glycosyltransferase essential for the synthesis of a bacterial adhesion protein

Chen

Seepersaud

Bensing

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

O-glycosylation of Ser and Thr residues is an important process in all organisms, which is only poorly understood. Such modification is required for the export and function of adhesin proteins that mediate the attachment of pathogenic Gram-positive bacteria to host cells. Here, we have analyzed the mechanism by which the cytosolic O-glycosyltransferase GtfA/B of Streptococcus gordonii modifies the Ser/Thr-rich repeats of adhesin. The enzyme is a tetramer containing two molecules each of GtfA and GtfB. The two subunits have the same fold, but only GtfA contains an active site, whereas GtfB provides the primary binding site for adhesin. During a first phase of glycosylation, the conformation of GtfB is restrained by GtfA to bind substrate with unmodified Ser/Thr residues. In a slow second phase, GtfB recognizes residues that are already modified with N-acetylglucosamine, likely by converting into a relaxed conformation in which one interface with GtfA is broken. These results explain how the glycosyltransferase modifies a progressively changing substrate molecule.

show abstract

Section: Discussionmentioning

confidence: 99%

Mechanism of a cytosolic O -glycosyltransferase essential for the synthesis of a bacterial adhesion protein

Chen

Seepersaud

Bensing

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Both steps occur via transition states with substantial oxocarbenium ion character (40,41). In family GH-16 glycoside hydrolases, the catalytic nucleophile and acid/base residues have been unambiguously identified in the P. macerans and B. licheniformis 1,3-1,4-␤-glucanases (21,22) and have been confirmed in the P. carrageenovora -carrageenase (24). Sequence and structure similarities between members of family GH-16 allowed the rapid identification of the catalytic machinery of the two ␤-agarases on strand ␤9 with Glu-147 and Glu-184 as the nucleophile and Glu-152 and Glu-189 as the acid/base in ␤-AgaA_CM and ␤-AgaB, respectively.…”

Section: Structural Comparison With Family Gh-16 Enzymes-mentioning

confidence: 99%

The Three-dimensional Structures of Two β-Agarases

Allouch

Jam

Helbert

et al. 2003

Journal of Biological Chemistry

129

132

View full text Add to dashboard Cite

Agars are important gelifying agents for biochemical use and the food industry. To cleave the ␤-1,4-linkages between ␤-D-galactose and ␣-L-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called ␤-agarases. ␤-Agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized. Here we report the first crystal structure of these two ␤-agarases. The two proteins were overproduced in Escherichia coli and crystallized, and the crystal structures were determined at 1.48 and 2.3 Å for ␤-agarases A and B, respectively. The structure of ␤-agarase A was solved by the multiple anomalous diffraction method, whereas ␤-agarase B was solved with molecular replacement using ␤-agarase A as model. Their structures adopt a jelly roll fold with a deep active site channel harboring the catalytic machinery, namely the nucleophilic residues Glu-147 and Glu-184 and the acid/ base residues Glu-152 and Glu-189 for ␤-agarases A and B, respectively. The structures of the agarases were compared with those of two lichenases and of a -carrageenase, which all belong to family 16 of the glycoside hydrolases in order to pinpoint the residues responsible for their widely differing substrate specificity. The relationship between structure and enzymatic activity of the two ␤-agarases from Z. galactanivorans Dsij was studied by analysis of the degradation products starting with different oligosaccharides. The combination of the structural and biochemical results allowed the determination of the number of subsites present in the catalytic cleft of the ␤-agarases.Agarose is a hydrophilic polysaccharide found in the cell wall of marine red algae (Rhodophyceaea) (1), where it naturally occurs in the form of a pseudocrystalline matrix associated with cellulose (2). It consists of a linear backbone of galactopyranose residues linked by alternating ␣-1,3-and ␤-1,4-linkages. Whereas the ␤-linked residues are in the D configuration, the ␣-1,3-linked galactose units are in the rare L configuration and are further modified by a 3,6-anhydro bridge (2, 3) (Fig. 1a). On the basis of x-ray fiber diffraction, optical rotation calculations, and solution gel transition, both a parallel double helix as well as a single helix structure for agarose have been proposed, where the individual polysaccharide chains have a left-handed 3-fold helix symmetry and a pitch of 1.90 nm (3, 4). Agarose forms thermo-reversible gels structured by aggregates of agarose chains. These gels exhibit unique rheological properties and are widely used as texturing agents for various applications in the food industry or as a common laboratory medium for chromatographic separation or bacterial colony growth (5).Agarases are the glycoside hydrolases (GH) 1 that hydrolyze agarose. The ␤-agarases and the ␣-agarases cleave the internal ␤-1,4-and ␣-1,3-linkage of agarose, respectively. ␤-Agarases produce agaro-oligosaccharides in the series homologous to neoagarobiose (O-3,6-anhydro-␣-L-galact...

show abstract

“…They show sequence similarities with fl-l,3-glucanases (laminarinases) but not with 1,3-1,4-fl-glucanases from plants [8]. The crystal structure of the 1,3-1,4-fl-glucanase of B. macerans (BGLM) and the closely related hybrid Bacillus enzyme H(A16-M) are known as well as circularly permuted variants thereof [9][10][11]. 1,3-1,4-fl-Glucanases belong to the jellyroll fl-sandwich-type proteins, where the sheets of the sandwich are curved and create a channel for substrate binding and cleavage.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of Bacillus licheniformis 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolase at 1.8 Å resolution

et al. 1995

Self Cite

View full text Add to dashboard Cite

The crystal structure of the 1,3-1,4-1~-D-glucan 4-glucanohydrolasoe from Bacillus licheniformis is solved at a resolution of 1.8 A and refined to R = 16.5%. The protein has a similar g-sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16-M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3-1,4-1~-nglucan 4-glucanohydrolases.

show abstract

Crystal Structure and Site-directed Mutagenesis of Bacillus macerans Endo-1,3 –1,4- β-glucanase

Cited by 113 publications

References 45 publications

Mechanism of a cytosolic O -glycosyltransferase essential for the synthesis of a bacterial adhesion protein

Mechanism of a cytosolic O -glycosyltransferase essential for the synthesis of a bacterial adhesion protein

The Three-dimensional Structures of Two β-Agarases

Crystal structure of Bacillus licheniformis 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolase at 1.8 Å resolution

Contact Info

Product

Resources

About