title: Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction.
abstract: AlgE2, AlgE4, and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyze the post-polymerization conversion of beta-D-mannuronic acid residues into alpha-L-guluronic acid residues. To study the kinetics and mode of action of these enzymes, homopolymeric mannuronan and other alginate samples with various composition were epimerized by letting the enzymatic reaction take place in an NMR tube. Series of 1H NMR spectra were recorded to obtain a time-resolved picture of the epimerization progress and the formation of specific monomer sequences. Starting from mannuronan, guluronic acid contents of up to 82% were introduced by the enzymes, and the product specificity, substrate selectivity, and reaction rates have been investigated. To obtain direct information of the GulA-block formation, similar experiments were performed using a 13C-1-enriched mannuronan as substrate. The NMR results were found to be in good agreement with data obtained by a radioisotope assay based on 3H-5-labeled substrates.
authors: Department of Biotechnology, Norwegian Biopolymer Laboratory, Norwegian University of Science and Technology, NTNU, Norway.
authors: Martin
authors: M
authors: Hartmann
authors: Anne Sissel
authors: AS
authors: Duun
authors: Sidsel
authors: S
authors: Markussen
authors: Hans
authors: H
authors: Grasdalen
authors: Svein
authors: S
authors: Valla
authors: Gudmund
authors: G
authors: Skjåk-Braek
mesh_terms: D000464:Alginates / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000737:chemistry / Q000378:metabolism*; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes; D020723:Glucuronic Acid; D006603:Hexuronic Acids / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011108:Polymers / Q000378:metabolism
chemical_list: D000464:Alginates; D002247:Carbon Isotopes; D006603:Hexuronic Acids; D011108:Polymers; D020723:Glucuronic Acid; C008324:mannuronic acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase