Biochemical analysis of the processive mechanism for epimerization of alginate by mannuronan C-5 epimerase AlgE4.
The enzymes mannuronan C-5 epimerases catalyse the in-chain epimerisation of beta-D-mannuronic acid to alpha-L-guluronic acid in the last step of alginate biosynthesis. The recombinant C-5 epimerase AlgE4, encoded by the soil bacteria Azotobacter vinelandii and expressed in Escherichia coli, exhibits a non-random mode of action when acting on mannuronan and alginates of various monomeric compositions. The observed residue sequence has been suggested previously to be due to either a preferred attack or a processive mode of action. Based on methodologies involving specific degrading enzymes, NMR, electrospray ionisation mass spectrometry and capillary electrophoresis we show here that on average 10 residues are epimerised for each enzyme-substrate encounter. A subsite model for the enzyme is analysed by the same methodology using native and 13C-labelled mannuronan oligomers as substrate for the AlgE4 epimerase. A hexameric oligomer is the minimum size to accommodate activity. For hexa-, hepta- and octameric substrates the third M residue from the non-reducing end is epimerised first.
Campa
Cristiana
C
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
Holtan
Synnøve
S
Nilsen
Nadra
N
Bjerkan
Tonje M
TM
Stokke
Bjørn T
BT
Skjåk-Braek
Gudmund
G
D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000378:metabolism*; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism*; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013379:Substrate Specificity
D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D011108:Polymers; D011994:Recombinant Proteins; C007896:guluronic acid; D020723:Glucuronic Acid; C008324:mannuronic acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase