Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus

Abstract

sp. CH1 and on the computer-aided model building of the tertiary structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. It was found that R52, H243, E246, W249, M251, W304, E308, H374, and M378 were absolutely required for the activity of RaCE. F114 and W303 (and possibly R377) also contributed to catalysis. These possible catalytic residues protruded into or near the active-site cleft surrounded by the inner -helices in the predicted (/) 6 core barrel structure of RaCE.

Biochemical Characterization of a Thermophilic Cellobiose 2-Epimerase from a Thermohalophilic Bacterium,Rhodothermus marinusJCM9785

“…All the amino acid residues important for catalysis, which have been confirmed by site-directed mutagenesis studies, 9,10) were completely conserved in RmCE. It is assumed that His259 and His390 of RmCE, corresponding to the putative catalytic His residues of RaCE, porcine AGE, and Anabaena AGE, [8][9][10] play roles as catalytic amino acid residues. Consistently with this assumption, the pK a value of side chain of His is about 6.3, and RmCE is active in a range of pH 6-9 but not on the acidic side, although it was fully stable at such pH values.…”

“…At present, the threedimensional structure of CE has not been elucidated, and the structure-function relationship of CE is not fully understood, although some important amino acid residues for catalysis have been reported. 10) In contrast to AGE, which acts on N-acetlymannosamine and Nacetlyglucosamine, all the CEs reported to date act on -1,4-linked oligosaccharides and are inert toward monosaccharides, including N-acetlyglucosamine and N-acetlymannosamine. This implies the existence of structural elements for the recognition of the -1,4-glucosidic linkage at the reducing end of substrate, chain-length of a substrate, and monomer component of the substrate.…”

“…9) Secondary structure prediction suggested that RaCE is also formed by an ð=Þ 6 barrel as AGEs, and two putative catalytic His residues are observed at the equivalent positions. 10) These conserved His residues of RaCE were confirmed to be essential for the catalytic activity by site-directed mutagenesis, 10) suggesting that CE and AGE share the essential structure of the active site, including catalytic amino acids.…”

Catalysis, Structures, and Applications of Carbohydrate Epimerases

“…11) The residues Arg 52, Phe114, His243, Glu246, Trp249, Trp303, Trp304, Glu308, His374 and Arg377 were determined to be required for activity by site directed mutagenesis, 12) as shown in Fig. 1.…”

“…21) The tertiary structure of Mn 2+ dependent The model structure of RaCE was constructed with porcine kidney AGE (PDB code 1fp3) as a template. 12) In the ribbon stereodiagram representation of an (α α)6 barrel core structure (red, α helices; cyan and green, β sheets and loops), the amino acid residues possibly involved in catalysis are shown in the CPK representation (upper and lower).…”