Abstract
Some probiotics including lactobacilli, colonize host animal cells by targeting glycosaminoglycans (GAGs), such as heparin, located in the extracellular matrix. Recent studies have shown that several lactic acid bacteria degrade GAGs. Here we show the structure/function relationship of Lacticaseibacillus rhamnosus 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (KduI) crucial for metabolism of unsaturated glucuronic acid produced through degradation of GAGs. Crystal structures of ligand-free and bound KduIs were determined by X-ray crystallography and the enzyme was found to consist of six identical subunits and adopt a β-helix as a basic scaffold. Ligands structurally similar to the substrate were bound to the cleft of each enzyme subunit. Several residues located in the cleft interacted with ligands through hydrogen bonds and/or C-C contacts. In addition to substrate analogs, a metal ion coordinated to four residues, His198, His200, Glu205, and His248, in the cleft, and the enzyme activity was significantly inhibited by a chelator, ethylenediaminetetraacetic acid. Site-directed mutants in Arg163, Ile165, Thr184, Thr194, His200, Arg203, Tyr207, Met262, and Tyr269 in the cleft exhibited little enzyme activity, indicating that these residues and the metal ion constituted an active site in the cleft. This is the first report on the active site structure of KduI based on the ligand-bound complex.