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

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)−4‐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′ hydroxyl and the C‐4′ 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.