Abstract
GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.
Highlights
The kinetic constants of this variant were very similar to those of the wild-type enzyme when using maltohexaose as the acceptor substrate (Table 1). Perhaps this is to be expected because the secondary binding site is quite remote from the donor and acceptor sites, and initial rates are measured in the enzyme assay when oligosaccharides have not yet reached significant lengths
The strain complemented with the GlgE variant grew normally and produced ␣-glucan in a manner similar to the wild-type strain (Fig. 7). This suggests that the secondary binding site is not essential in vivo in the conditions tested despite a potential loss of activity
The occupancy of the donor subsites Ϫ2 and Ϫ1 together with subsites ϩ1 to ϩ6 indicated that this represents a product-bound structure that reveals the locations of the first six acceptor subsites
Summary
Mapping the Acceptor Binding Site—There are two conspicuous clefts that are adjacent to the donor binding site (Fig. 2). By monitoring the production of inorganic phosphate with malachite green, the kinetics of each of the three variants was tested using maltohexaose as the acceptor. Little difference in the kinetics was observed compared with the wild-type enzyme (Table 1). It would seem that short linear acceptors do not bind in the linear cleft When these GlgE variants were assayed with ␣-glucan polymer isolated from Streptomyces venezuelae rather than maltohexaose, their values of kcaaptp/ Km app were less than 2-fold different from that of the wild-type enzyme (data not shown), suggesting that the linear cleft is not important in the accommodation of the larger acceptor either. Kinetics of GlgE variants with maltohexaose as the donor and 0.25 mM ␣-maltose 1-phosphate
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