title: Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-alpha-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli.
abstract: An Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1.8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57,184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequences identity was apparent with the 4-alpha-glucano-transferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated maIQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MaIP). The translation stop codon of MaIQ overlapped the translation start codon of the putative maIP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-alpha-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MaIQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves.
authors.affiliation: Department of Molecular Microbiology, Centre for Applied Microbiology and Research, Porton Down Salisbury SP4 OJG, UK.
authors.forename: Sayed K
authors.initials: SK
authors.lastname: Goda
authors.affiliation: Department of Biochemistry, University of Southampton, Bassett Crescent East, Southampton SO 16 7PX, UK.
authors.forename: Omima
authors.initials: O
authors.lastname: Eissa
authors.affiliation: Department of Biochemistry, University of Southampton, Bassett Crescent East, Southampton SO 16 7PX, UK.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
authors.affiliation: Department of Molecular Microbiology, Centre for Applied Microbiology and Research, Porton Down Salisbury SP4 OJG, UK.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D000595:Amino Acid Sequence; D000688:Amylose / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000235:genetics; D006004:Glycogen Debranching Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D006004:Glycogen Debranching Enzyme System; D011994:Recombinant Proteins; D000688:Amylose; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase; C020569:4 alpha-glucanotransferase