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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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Structural comparison of raffinose- and sucrose-bound E342A with apo wild-type and E342A levansucrase. (A) Stereo diagram of the superimposition of apo wild-type (green), E342A (grey) and raffinose-bound E342A (pale red). (B) Superposition of sucrose-bound (1PT2, [11], light blue) and raffinose-bound E342A (this study). Dashed lines in cyan indicate H-bond interactions conserved between both complexes, those in red indicate additional contacts made by the galactosyl moiety.
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Figure 3: Structural comparison of raffinose- and sucrose-bound E342A with apo wild-type and E342A levansucrase. (A) Stereo diagram of the superimposition of apo wild-type (green), E342A (grey) and raffinose-bound E342A (pale red). (B) Superposition of sucrose-bound (1PT2, [11], light blue) and raffinose-bound E342A (this study). Dashed lines in cyan indicate H-bond interactions conserved between both complexes, those in red indicate additional contacts made by the galactosyl moiety.

Mentions: In conclusion, the D247A and D86A mutations have little or no impact on the side chain configurations and interactions elsewhere in the active site, while the Glu342A substitution has knock-on effects for the network of non-covalent interactions around the Glu342 carboxylate. We note however, that binding of the donor substrate to levansucrase-E342A largely restores the side chain configuration of apo wild-type levansucrase (Figures 2D and 3A), prompting Arg246 and Arg360 to swing back into their original position.


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

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

Structural comparison of raffinose- and sucrose-bound E342A with apo wild-type and E342A levansucrase. (A) Stereo diagram of the superimposition of apo wild-type (green), E342A (grey) and raffinose-bound E342A (pale red). (B) Superposition of sucrose-bound (1PT2, [11], light blue) and raffinose-bound E342A (this study). Dashed lines in cyan indicate H-bond interactions conserved between both complexes, those in red indicate additional contacts made by the galactosyl moiety.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Structural comparison of raffinose- and sucrose-bound E342A with apo wild-type and E342A levansucrase. (A) Stereo diagram of the superimposition of apo wild-type (green), E342A (grey) and raffinose-bound E342A (pale red). (B) Superposition of sucrose-bound (1PT2, [11], light blue) and raffinose-bound E342A (this study). Dashed lines in cyan indicate H-bond interactions conserved between both complexes, those in red indicate additional contacts made by the galactosyl moiety.
Mentions: In conclusion, the D247A and D86A mutations have little or no impact on the side chain configurations and interactions elsewhere in the active site, while the Glu342A substitution has knock-on effects for the network of non-covalent interactions around the Glu342 carboxylate. We note however, that binding of the donor substrate to levansucrase-E342A largely restores the side chain configuration of apo wild-type levansucrase (Figures 2D and 3A), prompting Arg246 and Arg360 to swing back into their original position.

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
Related in: MedlinePlus