title: The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase.
abstract: A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmbeta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1-->4)-Glcp (maltose), alpha-Glcp-(1-->4)-GlcNp (maltosamine), alpha-Glcp-(1-->4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1-->4)-Manp, alpha-Glcp-(1-->4)-Xylp and alpha-Glcp-(1-->4)- L-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baumann
authors.forename: Bent O
authors.initials: BO
authors.lastname: Petersen
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Westphal
authors.forename: Henk
authors.initials: H
authors.lastname: Schols
authors.forename: Adiphol
authors.initials: A
authors.lastname: Dilokpimol
authors.forename: Maher A
authors.initials: MA
authors.lastname: Hachem
authors.forename: Sampo J
authors.initials: SJ
authors.lastname: Lahtinen
authors.forename: Jens Ø
authors.initials: JØ
authors.lastname: Duus
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001692:Biological Transport; D005960:Glucosides / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D007779:Lactobacillus acidophilus / Q000201:enzymology / Q000378:metabolism*; D008968:Molecular Conformation; D011134:Polysaccharides / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005960:Glucosides; D011134:Polysaccharides; C008315:maltodextrin; D005964:Glucosyltransferases; C021535:maltose phosphorylase