Acta Crystallographica Section D

Biological Crystallography

Volume 69, Part 4 (April 2013)


research papers



Acta Cryst. (2013). D69, 658-668    [ doi:10.1107/S0907444913001030 ]

Structure and in silico substrate-binding mode of ADP-L-glycero-D-manno-heptose 6-epimerase from Burkholderia thailandensis

M.-S. Kim, A. Lim, S. W. Yang, J. Park, D. Lee and D. H. Shin

Abstract: ADP-L-glycero-D-manno-heptose 6-epimerase (AGME), the product of the rfaD gene, is the last enzyme in the heptose-biosynthesis pathway; it converts ADP-D-glycero-D-manno-heptose (ADP-D,D-Hep) to ADP-L-glycero-D-manno-heptose (ADP-L,D-Hep). AGME contains a catalytic triad involved in catalyzing hydride transfer with the aid of NADP+. Defective lipopolysaccharide is found in bacterial mutants lacking this gene. Therefore, it is an interesting target enzyme for a novel epimerase inhibitor for use as a co-therapy with antibiotics. The crystal structure of AGME from Burkholderia thailandensis (BtAGME), a surrogate organism for studying the pathogenicity of melioidosis caused by B. pseudomallei, has been determined. The crystal structure determined with co-purified NADP+ revealed common as well as unique structural properties of the AGME family when compared with UDP-galactose 4-epimerase homologues. They form a similar architecture with conserved catalytic residues. Nevertheless, there are differences in the substrate- and cofactor-binding cavities and the oligomerization domains. Structural comparison of BtAGME with AGME from Escherichia coli indicates that they may recognize their substrate in a `lock-and-key' fashion. Unique structural features of BtAGME are found in two regions. The first region is the loop between [beta]8 and [beta]9, affecting the binding affinity of BtAGME for the ADP moiety of ADP-D,D-Hep. The second region is helix [alpha]8, which induces decamerization at low pH that is not found in other AGMEs. With the E210G mutant, it was observed that the resistance of the wild type to acid-induced denaturation is related to the decameric state. An in silico study was performed using the Surflex-Dock GeomX module of the SYBYL-X 1.3 software to predict the catalytic mechanism of BtAGME with its substrate, ADP-D,D-Hep. In the in silico study, the C7'' hydroxymethyl group of ADP-D,D-Hep is predicted to form hydrogen bonds to Ser116 and Gln293. With the aid of these interactions, the hydroxyl of Tyr139 forms a hydrogen bond to O6´´ of ADP-D,D-Hep and the proton at C6´´ orients closely to C4 of NADP+. Therefore, the in silico study supports a one-base mechanism as a major catalytic pathway, in which Tyr139 solely functions as a catalytic acid/base residue. These results provide a new insight into the development of an epimerase inhibitor as an antibiotic adjuvant against melioidosis.

PDB reference: 4ej0

Keywords: ADP-L-glycero-D-manno-heptose 6-epimerase; in silico study; ADP-D-glycero-D-manno-heptose; epimerases.


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