Abstract
Sucrose isomerase NX-5 from Erwinia rhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonas mesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.
Highlights
IntroductionIsomaltulose ( known as α-D-glucopyranosyl-1,6-Dfructofuranose or palatinose) and trehalulose (α-Dglucopyranosyl-1,1-D-fructofuranose) are two structural isomers of sucrose
Isomaltulose and trehalulose (α-Dglucopyranosyl-1,1-D-fructofuranose) are two structural isomers of sucrose
The isomaltulose synthase NX-5 used for the crystallographic study contained residues 1-600 and a Cterminal His-tag derived from the pET-22b expression vector
Summary
Isomaltulose ( known as α-D-glucopyranosyl-1,6-Dfructofuranose or palatinose) and trehalulose (α-Dglucopyranosyl-1,1-D-fructofuranose) are two structural isomers of sucrose. They possess similar physical and organoleptic properties as sucrose, they can prevent tooth decay and attenuate the glycemic index and insulin levels in the bloodstream [1,2,3,4,5]. They have been identified as valuable sucrose substitutes for obese and diabetic individuals [6]. Based on the product preference of sucrose isomers, SIases can be divided into two groups, isomaltulose synthases and trehalulose synthases.
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