Applying pairwise combinations of amino acid mutations for sorting out highly efficient glucosylation tools for chemo-enzymatic synthesis of bacterial oligosaccharides.
Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea , a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semirational sublibraries accounting in total for 20,000 variants, we report here the isolation of three double mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement toward both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor as compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.
Université de Toulouse, INSA,UPS,INP, LISBP, Toulouse, France.
Elise
E
Champion
Frédéric
F
Guérin
Claire
C
Moulis
Sophie
S
Barbe
Thu Hoai
TH
Tran
Sandrine
S
Morel
Karine
K
Descroix
Pierre
P
Monsan
Lionel
L
Mourey
Laurence A
LA
Mulard
Samuel
S
Tranier
Magali
M
Remaud-Siméon
Isabelle
I
André
D000596:Amino Acids / Q000235:genetics*; D055162:Biocatalysis; D004795:Enzyme Stability; D014644:Genetic Variation / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D009343:Neisseria / Q000201:enzymology*; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry
D000596:Amino Acids; D009844:Oligosaccharides; D005964:Glucosyltransferases; C019700:amylosucrase