Limits...
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.

Show MeSH

Related in: MedlinePlus

Superimposition of raffinose-bound complexes of T. maritima invertase and B. subtilis levansucrase. Raffinose-bound structures of T. maritima invertase (blue, 1W2T, [23]) and B. subtilis levansucrase-E342A (pale red) were superimposed by matching coordinates of three atoms of the raffinose ligand: fructosyl C2', C5' and glucosyl C5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2277421&req=5

Figure 5: Superimposition of raffinose-bound complexes of T. maritima invertase and B. subtilis levansucrase. Raffinose-bound structures of T. maritima invertase (blue, 1W2T, [23]) and B. subtilis levansucrase-E342A (pale red) were superimposed by matching coordinates of three atoms of the raffinose ligand: fructosyl C2', C5' and glucosyl C5.

Mentions: We present here the raffinose-bound complex of levansucrase-E342A in addition to the apo crystal structures of the three inactive point mutants D86A, D247A and E342A. Our previous study of the structure of B. subtilis levansucrase [11] established, based largely on structural arguments, the function of these three strictly conserved carboxylate side chains in the active site. The present raffinose complex reinforces a view that donor substrate recognition in B. subtilis levansucrase rests primarily on the common sucrosyl unit, whereas the galactosyl moiety, which protrudes out of the active site, makes only a few water-mediated H-bonds, pointing three unliganded hydroxyl groups to the bulk solvent. This mode of binding is echoed by the raffinose-bound complex of Thermotoga maritima invertase, which belongs to GH family 32 and which, like levansucrase, mediates hydrolysis of the glycosidic bond through a double displacement reaction mechanism [23]. In the latter study, an inert complex was facilitated by mutating the proton donor (Glu190) to aspartic acid. Superimposing the two complexes by matching the positions of 3 ligand atoms (Figure 5), reveals a very similar geometry of the ligand, and an almost perfect overlap of the catalytic residues. While there is significant variation of structural elements mediating specificity-determining contacts with the ligand, specific recognition of the outermost saccharide unit is weak in both structures and does not involve direct H-bonds. Nevertheless, the T. maritima complex includes notable van der Waals interactions between the galactose and Trp41, for which there is no counterpart in B. subtilis levansucrase (Figure 5). The importance of Glu340, Arg246 and Arg360 in forming specificity-determining contacts with the donor substrate is illustrated by the mutagenesis data obtained for levansucrase from Bacillus megaterium (74% identity on amino acid level). Mutating these side chains was reported as nearly abolishing hydrolase activity [24].


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

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

Superimposition of raffinose-bound complexes of T. maritima invertase and B. subtilis levansucrase. Raffinose-bound structures of T. maritima invertase (blue, 1W2T, [23]) and B. subtilis levansucrase-E342A (pale red) were superimposed by matching coordinates of three atoms of the raffinose ligand: fructosyl C2', C5' and glucosyl C5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Superimposition of raffinose-bound complexes of T. maritima invertase and B. subtilis levansucrase. Raffinose-bound structures of T. maritima invertase (blue, 1W2T, [23]) and B. subtilis levansucrase-E342A (pale red) were superimposed by matching coordinates of three atoms of the raffinose ligand: fructosyl C2', C5' and glucosyl C5.
Mentions: We present here the raffinose-bound complex of levansucrase-E342A in addition to the apo crystal structures of the three inactive point mutants D86A, D247A and E342A. Our previous study of the structure of B. subtilis levansucrase [11] established, based largely on structural arguments, the function of these three strictly conserved carboxylate side chains in the active site. The present raffinose complex reinforces a view that donor substrate recognition in B. subtilis levansucrase rests primarily on the common sucrosyl unit, whereas the galactosyl moiety, which protrudes out of the active site, makes only a few water-mediated H-bonds, pointing three unliganded hydroxyl groups to the bulk solvent. This mode of binding is echoed by the raffinose-bound complex of Thermotoga maritima invertase, which belongs to GH family 32 and which, like levansucrase, mediates hydrolysis of the glycosidic bond through a double displacement reaction mechanism [23]. In the latter study, an inert complex was facilitated by mutating the proton donor (Glu190) to aspartic acid. Superimposing the two complexes by matching the positions of 3 ligand atoms (Figure 5), reveals a very similar geometry of the ligand, and an almost perfect overlap of the catalytic residues. While there is significant variation of structural elements mediating specificity-determining contacts with the ligand, specific recognition of the outermost saccharide unit is weak in both structures and does not involve direct H-bonds. Nevertheless, the T. maritima complex includes notable van der Waals interactions between the galactose and Trp41, for which there is no counterpart in B. subtilis levansucrase (Figure 5). The importance of Glu340, Arg246 and Arg360 in forming specificity-determining contacts with the donor substrate is illustrated by the mutagenesis data obtained for levansucrase from Bacillus megaterium (74% identity on amino acid level). Mutating these side chains was reported as nearly abolishing hydrolase activity [24].

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