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d‐Allulose has potential as a low‐calorie sweetener which can suppress fat accumulation. Several enzymes capable of d‐allulose production have been isolated, including d‐tagatose 3‐epimerases. Here, we report the isolation of a novel protein from Methylomonas sp. expected to be a putative enzyme based on sequence similarity to ketose 3‐epimerase. The synthesized gene encoding the deduced ketose 3‐epimerase was expressed as a recombinant enzyme in Escherichia coli, and it exhibited the highest enzymatic activity toward l‐ribulose, followed by d‐ribulose and d‐allulose. The X‐ray structure analysis of l‐ribulose 3‐epimerase from Methylomonas sp. (MetLRE) revealed a homodimeric enzyme, the first reported structure of dimeric l‐ribulose 3‐epimerase. The monomeric structure of MetLRE is similar to that of homotetrameric l‐ribulose 3‐epimerases, but the short C‐terminal α‐helix of MetLRE is unique and different from those of known l‐ribulose 3 epimerases. The length of the C‐terminal α‐helix was thought to be involved in tetramerization and increasing stability; however, the addition of residues to MetLRE at the C terminus did not lead to tetramer formation. MetLRE is the first dimeric l‐ribulose 3‐epimerase identified to exhibit high relative activity toward d‐allulose., d‐Allulose has potential as a low‐calorie sweetener which can suppress fat accumulation. The enzymes capable of d‐allulose production have attracted attention. Here, we report the X‐ray structure of a novel l‐ribulose 3‐epimerase from Methylomonas sp. (MetLRE) which also has enzymatic activity towards d‐allulose, showing that MetLRE is the first dimeric l‐ribulose 3‐epimerase identified to exhibit high relative activity toward d‐allulose. ​

l-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. Previously determined X-ray structures of l-RhI showed a hydride-shift mechanism for the isomerization of substrates in a linear form, but the mechanism for opening of the sugar-ring is still unclear. To elucidate this mechanism, we determined X-ray structures of a mutant l-RhI in complex with l-rhamnopyranose and d-allopyranose. Results suggest that a catalytic water molecule, which acts as an acid/base catalyst in the isomerization reaction, is likely to be involved in pyranose-ring opening, and that a newly found substrate sub-binding site in the vicinity of the catalytic site may recognize different anomers of substrates., Highlights: ▸ l-Rhamnose isomerase catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. ▸ The catalytic reaction includes opening of the sugar-ring, but the mechanism is unclear. ▸ The structure of the enzyme with substrates in a pyranose ring form was determined. ▸ A catalytic water molecule is probably involved in opening the pyranose ring. ▸ A newly found substrate sub-binding site recognizes the anomers of substrates.