Crystal Structures of Lacticaseibacillus 4-Deoxy-L-threo-5-hexosulose-uronate Ketol-isomerase KduI in Complex with Substrate Analogs.

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 ß-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.

High-resolution crystal structures of the glycoside hydrolase family 45 endoglucanase EG27II from the snail Ampullaria crossean.

Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Šresolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double-ψ six-stranded ß-barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high-resolution structures of EG27II provide a large amount of information for structure-based enzyme modification and cell-surface engineering, which will advance biofuel production using animal-derived cellulases.