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Nakai, H, Petersen, BO, Westphal, Y, Dilokpimol, A, Abou Hachem, M, Duus, JØ, Schols, HA and Svensson, B (2010) Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase. Protein Eng. Des. Sel. 23:781-7
Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (alpha/alpha)(6)-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426-Ala431 in TP and Thr419-Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (alpha1, alpha1)- and alpha-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.
Amino Acid Substitution; Binding Sites; Biocatalysis; Disaccharides/chemistry; Disaccharides/genetics; Disaccharides/metabolism; Glucosyltransferases/chemistry; Glucosyltransferases/genetics; Glucosyltransferases/metabolism; Lactobacillus acidophilus/enzymology; Phosphorylases/chemistry; Phosphorylases/genetics; Phosphorylases/metabolism; Phosphorylation; Sequence Alignment; Substrate Specificity; Trehalose/chemistry; Trehalose/genetics; Trehalose/metabolism
|Gene product||Qualifier||GO Term||Evidence Code||with/from||Aspect||Extension||Notes||Status|
|GO:0050082: maltose phosphorylase activity||
Fig. 2 shows a short loop 3, corresponding to LaMP His413–Glu415 in the (α/α)6-barrel catalytic domain of GH65 disaccharide phosphorylase, is identified to be a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis. Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (α/α)6-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with β-glucose 1-phosphate and glucose as donor and acceptor, respectively.
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