Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase.
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.
Nakai
Hiroyuki
H
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.
Petersen
Bent O
BO
Westphal
Yvonne
Y
Dilokpimol
Adiphol
A
Abou Hachem
Maher
M
Duus
Jens Ø
JØ
Schols
Henk A
HA
Svensson
Birte
B
D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D004187:Disaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D016415:Sequence Alignment; D013379:Substrate Specificity; D014199:Trehalose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004187:Disaccharides; C008237:kojibiose; D014199:Trehalose; D005964:Glucosyltransferases; D006005:Phosphorylases; C021535:maltose phosphorylase