The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase.
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.
Nakai
Hiroyuki
H
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
Baumann
Martin J
MJ
Petersen
Bent O
BO
Westphal
Yvonne
Y
Schols
Henk
H
Dilokpimol
Adiphol
A
Hachem
Maher A
MA
Lahtinen
Sampo J
SJ
Duus
Jens Ø
JØ
Svensson
Birte
B
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
D005960:Glucosides; D011134:Polysaccharides; C008315:maltodextrin; D005964:Glucosyltransferases; C021535:maltose phosphorylase