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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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The 5-bladed β-propeller fold of the catalytic domain of glycoside hydrolase families 68 and 32. Ribbon diagram of B. subtilis levansucrase in complex with raffinose (shown as stick model).
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Figure 1: The 5-bladed β-propeller fold of the catalytic domain of glycoside hydrolase families 68 and 32. Ribbon diagram of B. subtilis levansucrase in complex with raffinose (shown as stick model).

Mentions: We recently determined the crystal structures of B. subtilis levansucrase in the ligand-free form and bound to the fructosyl donor substrate sucrose [11]. Our structures established that the catalytic domain of GH family 68 enzymes folds into a 5-bladed β-propeller with the active site located in a deep axial pocket (Figure 1). This fold is shared by the catalytic domain of GH family 32 of retaining enzymes [12-15], as well as by the distantly related family 43 of inverting glycoside hydrolases [16,17] (see also reference [18] for a review of structure-function relationships in levansucrases). In agreement with a rich body of biochemical data [8,19-22], we proposed that the strictly conserved Asp86 (nucleophile) and Glu342 (proton donor) represent the two canonical catalytic carboxylate groups, while a third invariant carboxylate, Asp247, may aid catalysis by stabilizing the transition state of the oxocarbenium ion by forming close hydrogen bond contacts with two of the fructosyl hydroxyls [11]. While this assignment was confirmed in subsequent structural and biochemical studies of several ortho- and paralogs [12-15,23-25], the characterization of the structural determinants of levan synthesis has remained incomplete. In particular, the mode of and elements required for acceptor substrate binding remain unclear.


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

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

The 5-bladed β-propeller fold of the catalytic domain of glycoside hydrolase families 68 and 32. Ribbon diagram of B. subtilis levansucrase in complex with raffinose (shown as stick model).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The 5-bladed β-propeller fold of the catalytic domain of glycoside hydrolase families 68 and 32. Ribbon diagram of B. subtilis levansucrase in complex with raffinose (shown as stick model).
Mentions: We recently determined the crystal structures of B. subtilis levansucrase in the ligand-free form and bound to the fructosyl donor substrate sucrose [11]. Our structures established that the catalytic domain of GH family 68 enzymes folds into a 5-bladed β-propeller with the active site located in a deep axial pocket (Figure 1). This fold is shared by the catalytic domain of GH family 32 of retaining enzymes [12-15], as well as by the distantly related family 43 of inverting glycoside hydrolases [16,17] (see also reference [18] for a review of structure-function relationships in levansucrases). In agreement with a rich body of biochemical data [8,19-22], we proposed that the strictly conserved Asp86 (nucleophile) and Glu342 (proton donor) represent the two canonical catalytic carboxylate groups, while a third invariant carboxylate, Asp247, may aid catalysis by stabilizing the transition state of the oxocarbenium ion by forming close hydrogen bond contacts with two of the fructosyl hydroxyls [11]. While this assignment was confirmed in subsequent structural and biochemical studies of several ortho- and paralogs [12-15,23-25], the characterization of the structural determinants of levan synthesis has remained incomplete. In particular, the mode of and elements required for acceptor substrate binding remain unclear.

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