The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity

Abstract Pseudomonas stutzeri l -rhamnose isomerase ( P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli ( E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with l -rhamnose and d -allose which has different configurations of C4 and C5 from l -rhamnose, were determined at a resolution of 2.0 A, 1.97 A, and 1.97 A, respectively. P. stutzeri L-RhI has a large domain with a (β/α) 8 barrel fold and an additional small domain composed of seven α-helices, forming a homo tetramer, as found in E. coli L-RhI and d -xylose isomerases (D-XIs) from various microorganisms. The β1–α1 loop (Gly60–Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding β1–α1 loop is extended (Asp52–Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with l -rhamnose and d -allose show that both substrates are nicely fitted to the substrate -binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the β1–α1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of l -rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.