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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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Views of the active site of catalytically inactive mutants of B. subtilis levansucrase. Difference electron density maps were calculated with Fourier coefficients (Fo,mutant - Fo,wild-type) and model phases derived from the wild-type enzyme structure (1OYG, [11]). The maps (2.1 Å resolution) are contoured at 5σ (panels A-C) or 4σ (panel D), with positive and negative density in blue and red, respectively. The stick models are colour-coded by carbon atoms as follows: wild type enzyme (green), D86A (magenta – panel A), D247A (yellow – panel B), E342A (grey – panel C) and raffinose-bound E342A (pale red – panel D).
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Figure 2: Views of the active site of catalytically inactive mutants of B. subtilis levansucrase. Difference electron density maps were calculated with Fourier coefficients (Fo,mutant - Fo,wild-type) and model phases derived from the wild-type enzyme structure (1OYG, [11]). The maps (2.1 Å resolution) are contoured at 5σ (panels A-C) or 4σ (panel D), with positive and negative density in blue and red, respectively. The stick models are colour-coded by carbon atoms as follows: wild type enzyme (green), D86A (magenta – panel A), D247A (yellow – panel B), E342A (grey – panel C) and raffinose-bound E342A (pale red – panel D).

Mentions: Crystals of the inactive single site mutants D86A, D247A and E342A of B. subtilis levansucrase grew at similar solution conditions as the wild-type enzyme. Crystals were in space group P212121, diffracting to beyond 2.0 Å. Geometric constraints of the detector, rather than crystal quality limited data acquisition on the home source to a maximum resolution of 2.1 Å (Table 1). The diffraction data of mutant-forms of levansucrase were highly isomorphous to those of crystal form I of wild-type levansucrase (cf. [11]). Prior to structure (re-)building and refinement, structural differences were ascertained by way of difference Fourier maps (Figure 2), and the structures were refined starting from the wild-type model, altering the mutation sites to alanine (Table 2). The backbone structures superimpose closely with that of the wild-type enzyme. The root mean square deviation (RMSD) for 1760 backbone atoms between the mutant and wild type structures vary from 0.11 Å (D86A, E342A) to 0.12 Å (D247A). The RMSD for 1717 side chain atoms between mutants and wild type structures are 0.5 Å or less (0.33 Å – D86A, 0.36 Å – D247A, 0.48 Å – E342A), corresponding to ~2 times the estimated coordinate error at 2.1 Å. This indicates that overall the mutations induce only minimal structural changes. The missing carboxylate groups in the D86A and D247A mutants are clearly marked by negative density in the difference Fourier maps, but result in no other notable changes in the active site (Figures 2A and 2B).


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

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

Views of the active site of catalytically inactive mutants of B. subtilis levansucrase. Difference electron density maps were calculated with Fourier coefficients (Fo,mutant - Fo,wild-type) and model phases derived from the wild-type enzyme structure (1OYG, [11]). The maps (2.1 Å resolution) are contoured at 5σ (panels A-C) or 4σ (panel D), with positive and negative density in blue and red, respectively. The stick models are colour-coded by carbon atoms as follows: wild type enzyme (green), D86A (magenta – panel A), D247A (yellow – panel B), E342A (grey – panel C) and raffinose-bound E342A (pale red – panel D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Views of the active site of catalytically inactive mutants of B. subtilis levansucrase. Difference electron density maps were calculated with Fourier coefficients (Fo,mutant - Fo,wild-type) and model phases derived from the wild-type enzyme structure (1OYG, [11]). The maps (2.1 Å resolution) are contoured at 5σ (panels A-C) or 4σ (panel D), with positive and negative density in blue and red, respectively. The stick models are colour-coded by carbon atoms as follows: wild type enzyme (green), D86A (magenta – panel A), D247A (yellow – panel B), E342A (grey – panel C) and raffinose-bound E342A (pale red – panel D).
Mentions: Crystals of the inactive single site mutants D86A, D247A and E342A of B. subtilis levansucrase grew at similar solution conditions as the wild-type enzyme. Crystals were in space group P212121, diffracting to beyond 2.0 Å. Geometric constraints of the detector, rather than crystal quality limited data acquisition on the home source to a maximum resolution of 2.1 Å (Table 1). The diffraction data of mutant-forms of levansucrase were highly isomorphous to those of crystal form I of wild-type levansucrase (cf. [11]). Prior to structure (re-)building and refinement, structural differences were ascertained by way of difference Fourier maps (Figure 2), and the structures were refined starting from the wild-type model, altering the mutation sites to alanine (Table 2). The backbone structures superimpose closely with that of the wild-type enzyme. The root mean square deviation (RMSD) for 1760 backbone atoms between the mutant and wild type structures vary from 0.11 Å (D86A, E342A) to 0.12 Å (D247A). The RMSD for 1717 side chain atoms between mutants and wild type structures are 0.5 Å or less (0.33 Å – D86A, 0.36 Å – D247A, 0.48 Å – E342A), corresponding to ~2 times the estimated coordinate error at 2.1 Å. This indicates that overall the mutations induce only minimal structural changes. The missing carboxylate groups in the D86A and D247A mutants are clearly marked by negative density in the difference Fourier maps, but result in no other notable changes in the active site (Figures 2A and 2B).

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