Abstract
Carbohydrate-active enzymes (CAZymes) regulate the synthesis, degradation, and modification of the poly—and oligosaccharides in all three kingdoms of life. A novel carbohydrate acetylesterase from Sinorhizobium meliloti, designated SmAcE1, was identified, characterized, and crystallized. This SmAcE1 is classified into the carbohydrate esterase family 3 (CE3) based on the sequence alignments with other currently known carbohydrate esterase (CE) family enzymes. The SmAcE1 was crystallized as a hexamer in a space group P212121 with the unit cell parameters: a = 99.12 Å, b = 148.88 Å, c = 149.84 Å, and α = β = γ = 90.00°. The diffraction data set was collected up to a 2.05 Å resolution. Hydrolysis activity of SmAcE1 towards glucose pentaacetate and cellulose acetate was further confirmed using acetic acid release assay. Further crystallographic and functional analyses studies on SmAcE1 would be followed to fully understand the reaction mechanisms of CEs.
Highlights
Carbohydrate active enzymes (CAZymes) synthesize, degrade, and modify poly—or oligo saccharide chain, including cell wall in plant, and in bacteria and fungi [1,2,3,4]
In this study, based on the sequence identity with other known carbohydrate esterase family 3 (CE3) family enzymes, we identified a novel carbohydrate acetylesterase (SmAcE1) in CE3 family from Sinorhizobium meliloti, a nitrogen-fixing soil bacterium
Escherichia coli XL1-Blue (Stratagene, La Jolla, CA, USA) harboring pQE30-SmAcE1 was cultured at 310 K until the OD600 reached 0.5–0.7
Summary
Carbohydrate active enzymes (CAZymes) synthesize, degrade, and modify poly—or oligo saccharide chain, including cell wall in plant, and in bacteria and fungi [1,2,3,4] Among these proteins, carbohydrate esterases (CEs) play key roles in making other CAZymes access their carbohydrate substrates by removing acetyl groups from substituted saccharides with various chain lengths [5,6]. It is necessary to execute structural and biochemical investigations of bacterial CEs in order to understand their reaction mechanism as well as substrate specificity, which will eventually contribute to full understanding of their physiological roles as well as their biotechnological applications. We further characterized and crystallized the recombinant SmAcE1, which will pave a way for understanding of the catalytic mechanism as well as structural features of CE family members
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