Cloning and expression of three new Aazotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases.
The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5'-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5' part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.
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UNIGEN Center for Molecular Biology, Norwegian University of Science and Technology, N-7005 Trondheim, Norway.
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D000464:Alginates / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D005810:Multigene Family; D017386:Sequence Homology, Amino Acid
D000464:Alginates; D017931:DNA Primers; D004269:DNA, Bacterial; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase