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Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase Bacillus subtilis levansucrase.

Meng G, Fütterer K - BMC Struct. Biol. (2008)

Bottom Line: The D86A and D247A substitutions have little effect on the active site geometry.The raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety.The present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of Arg360 is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby Glu340 and Asn242 at the rim of a central pocket forming the active site.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK. g.meng@mail.cryst.bbk.ac.uk

ABSTRACT

Background: Fructans - beta-D-fructofuranosyl polymers with a sucrose starter unit - constitute a carbohydrate reservoir synthesised by a considerable number of bacteria and plant species. Biosynthesis of levan (alphaGlc(1-2)betaFru [(2-6)betaFru]n), an abundant form of bacterial fructan, is catalysed by levansucrase (sucrose:2,6-beta-D-fructan-6-beta-D-fructosyl transferase), utilizing sucrose as the sole substrate. Previously, we described the tertiary structure of Bacillus subtilis levansucrase in the ligand-free and sucrose-bound forms, establishing the mechanistic roles of three invariant carboxylate side chains, Asp86, Asp247 and Glu342, which are central to the double displacement reaction mechanism of fructosyl transfer. Still, the structural determinants of the fructosyl transfer reaction thus far have been only partially defined.

Results: Here, we report high-resolution structures of three levansucrase point mutants, D86A, D247A, and E342A, and that of raffinose-bound levansucrase-E342A. The D86A and D247A substitutions have little effect on the active site geometry. In marked contrast, the E342A mutant reveals conformational flexibility of functionally relevant side chains in the vicinity of the general acid Glu342, including Arg360, a residue required for levan polymerisation. The raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety.

Conclusion: The present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of Arg360 is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby Glu340 and Asn242 at the rim of a central pocket forming the active site.

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Schematic diagram of interactions between raffinose and levansucrase E342A. Interactions were calculated using LIGPLOT [41]. H-bond interactions, with distances in Å units, are indicated by dashed green lines. Residues making van der Waals or hydrophobic contacts are indicated by the 'bent comb' symbol. Water molecules appear as spheres in light blue, carbon, oxygen and nitrogen are in black, red and dark blue, respectively.
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Figure 4: Schematic diagram of interactions between raffinose and levansucrase E342A. Interactions were calculated using LIGPLOT [41]. H-bond interactions, with distances in Å units, are indicated by dashed green lines. Residues making van der Waals or hydrophobic contacts are indicated by the 'bent comb' symbol. Water molecules appear as spheres in light blue, carbon, oxygen and nitrogen are in black, red and dark blue, respectively.

Mentions: Following the terminology defined by Davies et al. [27], the active site of glycoside hydrolases can be divided into subsites with respect to the cleaved glycosidic bond. Applied to the present raffinose complex, subsite -1 coincides with the fructose, and subsites +1 and +2 with the glucose and galactose moieties, respectively (Figure 3). It is apparent that, with the exception of Arg246 and perhaps Glu342, side chains tend to form specificity-determining contacts with only one of the three subsites (Figures 3B and 4): the fructosyl moiety makes specificity-determining contacts with the side chains of Trp85 (3.1 Å to O6'), Asp86 (2.7 Å to O1'), Arg246 (3.2 Å to O3'), Asp247 (2.6 Å to O3' and 2.7 Å to O4'), and Glu342 (2.4 Å to O2' – assuming that the side chain conformation of Glu342 in the substrate-bound wild-type enzyme is identical to that of the ligand-free form). In subsite +1, the 2-, 3- and 4-hydroxyls of the glucosyl moiety form tight H-bond contacts (2.6 – 3.1 Å) with Arg360 and Glu340. These specificity-determining contacts lock the fructosyl and glucosyl units into defined orientations, positioning the anomeric carbon of the fructosyl unit within 3.2 Å of the nucleophile Asp86, and the glycosidic oxygen in close proximity (~2.5 Å) to the carboxylate of Glu342. In contrast, the galactosyl unit makes only few, water-mediated H-bonds, limited to the 6"-hydroxyl, with Asn242 and Tyr237, whereas the 2"-, 3"- and 4"-hydroxyl groups point into solvent (Figures 3A and 4).


Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase Bacillus subtilis levansucrase.

Meng G, Fütterer K - BMC Struct. Biol. (2008)

Schematic diagram of interactions between raffinose and levansucrase E342A. Interactions were calculated using LIGPLOT [41]. H-bond interactions, with distances in Å units, are indicated by dashed green lines. Residues making van der Waals or hydrophobic contacts are indicated by the 'bent comb' symbol. Water molecules appear as spheres in light blue, carbon, oxygen and nitrogen are in black, red and dark blue, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2277421&req=5

Figure 4: Schematic diagram of interactions between raffinose and levansucrase E342A. Interactions were calculated using LIGPLOT [41]. H-bond interactions, with distances in Å units, are indicated by dashed green lines. Residues making van der Waals or hydrophobic contacts are indicated by the 'bent comb' symbol. Water molecules appear as spheres in light blue, carbon, oxygen and nitrogen are in black, red and dark blue, respectively.
Mentions: Following the terminology defined by Davies et al. [27], the active site of glycoside hydrolases can be divided into subsites with respect to the cleaved glycosidic bond. Applied to the present raffinose complex, subsite -1 coincides with the fructose, and subsites +1 and +2 with the glucose and galactose moieties, respectively (Figure 3). It is apparent that, with the exception of Arg246 and perhaps Glu342, side chains tend to form specificity-determining contacts with only one of the three subsites (Figures 3B and 4): the fructosyl moiety makes specificity-determining contacts with the side chains of Trp85 (3.1 Å to O6'), Asp86 (2.7 Å to O1'), Arg246 (3.2 Å to O3'), Asp247 (2.6 Å to O3' and 2.7 Å to O4'), and Glu342 (2.4 Å to O2' – assuming that the side chain conformation of Glu342 in the substrate-bound wild-type enzyme is identical to that of the ligand-free form). In subsite +1, the 2-, 3- and 4-hydroxyls of the glucosyl moiety form tight H-bond contacts (2.6 – 3.1 Å) with Arg360 and Glu340. These specificity-determining contacts lock the fructosyl and glucosyl units into defined orientations, positioning the anomeric carbon of the fructosyl unit within 3.2 Å of the nucleophile Asp86, and the glycosidic oxygen in close proximity (~2.5 Å) to the carboxylate of Glu342. In contrast, the galactosyl unit makes only few, water-mediated H-bonds, limited to the 6"-hydroxyl, with Asn242 and Tyr237, whereas the 2"-, 3"- and 4"-hydroxyl groups point into solvent (Figures 3A and 4).

Bottom Line: The D86A and D247A substitutions have little effect on the active site geometry.The raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety.The present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of Arg360 is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby Glu340 and Asn242 at the rim of a central pocket forming the active site.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK. g.meng@mail.cryst.bbk.ac.uk

ABSTRACT

Background: Fructans - beta-D-fructofuranosyl polymers with a sucrose starter unit - constitute a carbohydrate reservoir synthesised by a considerable number of bacteria and plant species. Biosynthesis of levan (alphaGlc(1-2)betaFru [(2-6)betaFru]n), an abundant form of bacterial fructan, is catalysed by levansucrase (sucrose:2,6-beta-D-fructan-6-beta-D-fructosyl transferase), utilizing sucrose as the sole substrate. Previously, we described the tertiary structure of Bacillus subtilis levansucrase in the ligand-free and sucrose-bound forms, establishing the mechanistic roles of three invariant carboxylate side chains, Asp86, Asp247 and Glu342, which are central to the double displacement reaction mechanism of fructosyl transfer. Still, the structural determinants of the fructosyl transfer reaction thus far have been only partially defined.

Results: Here, we report high-resolution structures of three levansucrase point mutants, D86A, D247A, and E342A, and that of raffinose-bound levansucrase-E342A. The D86A and D247A substitutions have little effect on the active site geometry. In marked contrast, the E342A mutant reveals conformational flexibility of functionally relevant side chains in the vicinity of the general acid Glu342, including Arg360, a residue required for levan polymerisation. The raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety.

Conclusion: The present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of Arg360 is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby Glu340 and Asn242 at the rim of a central pocket forming the active site.

Show MeSH