Journal article
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 (alpha 1, alpha 1)- 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.
Language: | English |
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Publisher: | Oxford University Press |
Year: | 2010 |
Pages: | 781-787 |
ISSN: | 17410134 and 17410126 |
Types: | Journal article |
DOI: | 10.1093/protein/gzq055 |
ORCIDs: | Abou Hachem, Maher , Duus, Jens Øllgaard and Svensson, Birte |
Lactobacillus acidophilus NCFM glycoside hydrolase family 65 maltose phosphorylase reverse phosphorolysis specificity engineering
Amino Acid Substitution Binding Sites Biocatalysis Disaccharides Glucosyltransferases Lactobacillus acidophilus NCFM/maltose Phosphorylases Phosphorylation Sequence Alignment Substrate Specificity Trehalose family 65/Lactobacillus acidophilus glycoside hydrolase kojibiose phosphorolysis/specificity engineering phosphorylase/reverse