title: Combinatorial engineering of dextransucrase specificity.
abstract: We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.10(4) clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide (460)DYVHT(464) in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity.
authors.affiliation: Université de Toulouse; INSA, UPS, INP, LISBP, Toulouse, France ; CNRS, UMR5504, Toulouse, France ; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Irague
authors.forename: Laurence
authors.initials: L
authors.lastname: Tarquis
authors.forename: Isabelle
authors.initials: I
authors.lastname: André
authors.forename: Claire
authors.initials: C
authors.lastname: Moulis
authors.forename: Sandrine
authors.initials: S
authors.lastname: Morel
authors.forename: Pierre
authors.initials: P
authors.lastname: Monsan
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Potocki-Véronèse
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Siméon
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003911:Dextrans / Q000378:metabolism; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D003911:Dextrans; D005936:Glucans; D010446:Peptide Fragments; D019151:Peptide Library; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C033837:alternansucrase; C020025:dextransucrase