The molecular architecture of QdtA, a sugar 3,4‐ketoisomerase from Thermoanaerobacterium thermosaccharolyticum

Abstract

Unusual di-and trideoxysugars are often found on the O-antigens of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on various natural products. One such sugar is 3-acetamido-3,6-dideoxy-D-glucose. A key step in its biosynthesis, catalyzed by a 3,4-ketoisomerase, is the conversion of thymidine diphosphate (dTDP)24-keto-6-deoxyglucose to dTDP-3-keto-6-deoxyglucose. Here we report an X-ray analysis of a 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. For this investigation, the wild-type enzyme, referred to as QdtA, was crystallized in the presence of dTDP and its structure solved to 2.0-Å resolution. The dimeric enzyme adopts a three-dimensional architecture that is characteristic for proteins belonging to the cupin superfamily. In order to trap the dTDP-4-keto-6-deoxyglucose substrate into the active site, a mutant protein, H51N, was subsequently constructed, and the structure of this protein in complex with the dTDP-sugar ligand was solved to 1.9-Å resolution. Taken together, the structures suggest that His 51 serves as a catalytic base, that Tyr 37 likely functions as a catalytic acid, and that His 53 provides a proton shuttle between the C-3 0 hydroxyl and the C-4 0 keto group of the hexose. This study reports the first three-dimensional structure of a 3,4-ketoisomerase in complex with its dTDP-sugar substrate and thus sheds new molecular insight into this fascinating class of enzymes.

Bacterial Sugar 3,4-Ketoisomerases: Structural Insight into Product Stereochemistry

“…7 Iterative cycles of model building with COOT and refinement with REFMAC reduced the R work and R free to 20.5% and 23.7%, respectively, from 50–1.8 Å resolution.…”

“…In order to trap a dTDP-sugar in the active site, the H51N variant was employed for crystallizations. 7 …”

“…7 Given that QdtA and FdtA demonstrate an amino acid sequence identity of 55%, it was not surprising that QdtA adopted a similar cupin-type architecture. Importantly, however, it was possible to trap the dTDP-4-keto-6-deoxyglucose substrate into the QdtA active site by utilizing the catalytically inert H51N site-directed mutant variant for crystallizations.…”

Investigation of the enzymes required for the biosynthesis of an unusual formylated sugar in the emerging human pathogen Helicobacter canadensis

“…25 Until now, those sugar ketoisomerases belonging to the cupin superfamily were thought to function as dimers. [26][27][28][29][30] Interestingly, the two H. canadensis subunits in the asymmetric unit did not display the typical dimeric quaternary structure. Upon inspection of the crystalline packing interactions, it became clear that the H. canadensis ketoisomerase adopted a tetrameric quaternary structure with D2 symmetry.…”

Structure of the Bacillus anthracis dTDP-L-rhamnose-biosynthetic enzyme dTDP-4-dehydrorhamnose 3,5-epimerase (RfbC)

“…RmlC-like epimerases belong to the diverse cupin superfamily, which includes phosphomannose isomerases, transcription factors and storage proteins (Dunwell et al, 2001). Along with the related 3,4ketoisomerases, they are responsible for much of the diversity of the final products in various biosynthetic pathways that share the first two steps of the dTDP-l-rhamnose-biosynthetic pathway (Giraud et al, 2000;Thoden & Holden, 2014;Salinger et al, 2015;Dong et al, 2003;Kubiak et al, 2012). Here, we report the crystal structure of RfbC from B. anthracis determined at 1.63 Å resolution in complex with dTDP and pyrophosphate.…”