title: Properties of BoAGE2, a second N-acetyl-D-glucosamine 2-epimerase from Bacteroides ovatus ATCC 8483.
abstract: N-Acyl-D-Glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc). Bacteroides ovatus ATCC 8483 shows 3 putative genes for AGE activity (BACOVA_00274, BACOVA_01795 and BACOVA_01816). The BACOVA_00274 gene encodes an AGE (BoAGE1) with strong similarity to the AGE previously characterized in Bacteroides fragilis. Interestingly, the BACOVA_01816 gene (BoAGE2) shares 57% identity with Anabaena sp. CH1 AGE, but has an extra 27-amino acid tag sequence in the N-terminal. When cloned and expressed in Escherichia coli Rosetta (DE3)pLys, BACOVA_01816 was able to convert ManNAc into GlcNAc and vice versa. It was stable over a broad range of pHs and its activity was enhanced by ATP (20 μM). The incubation with ATP stabilized its structure, raising its melting temperature by about 8 °C. In addition, the catalytic efficiency for ManNAc synthesis was higher than that for GlcNAc synthesis. These characteristics make BoAGE2 a promising biocatalyst for sialic acid production using cheap GlcNAc as starting material. BoAGE2 could be considered a Renin-binding Protein and its interaction with renin was studied for the first time in a prokaryotic AGE. Surprisingly, renin activated BoAGE2, an effect which is contrary to that described for mammalian AGE and unrelated with the unique N-terminal tag, since a mutant without this tag was also activated by renin. When BoAGE2 sequence was compared with other related (real and putative) AGE described in the databases, it was seen that AGE enzymes can be divided in 3 different groups. The relationship between these groups is also discussed.
authors: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.
authors: Agustín
authors: A
authors: Sola-Carvajal
authors: Guiomar
authors: G
authors: Sánchez-Carrón
authors: María Inmaculada
authors: MI
authors: García-García
authors: Francisco
authors: F
authors: García-Carmona
authors: Álvaro
authors: Á
authors: Sánchez-Ferrer
mesh_terms: D000595:Amino Acid Sequence; D001439:Bacteroides / Q000201:enzymology*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012083:Renin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D012083:Renin; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase