A new Azotobacter vinelandii mannuronan C-5-epimerase gene (algG) is part of an alg gene cluster physically organized in a manner similar to that in Pseudomonas aeruginosa.
Alginate is an unbranched polysaccharide composed of the two sugar residues beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). The M/G ratio and sequence distribution in alginates vary and are of both biological and commercial significance. We have previously shown that a family of highly related mannuronan C-5-epimerase genes (algE1 to -E5) controls these parameters in Azotobacter vinelandii, by catalyzing the Ca2+-dependent conversion of M to G at the polymer level. In this report, we describe the cloning and expression of a new A. vinelandii epimerase gene (here designated algG), localized 29 nucleotides downstream of the previously described gene algJ. Sequence alignments show that algG does not belong to the same class of genes as algE1 to -E5 but that it shares 66% sequence identity with a previously described mannuronan C-5-epimerase gene (also designated algG) from Pseudomonas aeruginosa. A. vinelandii algG was expressed in Escherichia coli, and the enzyme was found to catalyze epimerization in the absence of Ca2+, although the presence of the cation stimulated the activity moderately. Surprisingly, all activity was blocked by Zn2+. P. aeruginosa AlgG has been reported to contain an N-terminal export signal sequence which is cleaved off during expression in E. coli. This does not happen with A. vinelandii AlgG, which appears to be produced at least partly in an insoluble form when expressed at high levels in E. coli. DNA sequencing analyses of the regions flanking algG suggest that the gene is localized in a cluster of genes putatively involved in alginate biosynthesis, and the organization of this cluster appears to be the same as previously described for P. aeruginosa.
Rehm
B H
BH
Lehrstuhl für Mikrobiologie der Mikroorganismen, Ruhr-Universität Bochum, Germany.
Ertesvåg
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Valla
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D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015032:Zinc / Q000494:pharmacology
D000464:Alginates; D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase; D015032:Zinc; D002118:Calcium