Combinatorial engineering of dextransucrase specificity

• Published in: PloS one 2013-10, Vol.8 (10), p.e77837
• Main Authors: Irague, Romain, Tarquis, Laurence, André, Isabelle, Moulis, Claire, Morel, Sandrine, Monsan, Pierre, Potocki-Véronèse, Gabrielle, Remaud-Siméon, Magali
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Automation; Bacteria; Catalysis; Chemical synthesis; Cloning; Combinatorial analysis; Dextrans - metabolism; Dextransucrase; Engineering; Enzymes; Glucans; Glucans - metabolism; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Glycoside hydrolase; Glycosyltransferases - metabolism; Hydrolase; Lactobacillus reuteri; Leuconostoc - enzymology; Leuconostoc - genetics; Leuconostoc mesenteroides; Libraries; Life Sciences; Linkages; Magnetic Resonance Spectroscopy; Medical screening; Models, Molecular; Molecular modelling; Molecular Sequence Data; Molecular weight; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Mutation - genetics; Peptide Fragments - metabolism; Peptide Library; Protein Engineering; Proteins; Saturation mutagenesis; Sequence Homology, Amino Acid; Structural analysis; Structure-Activity Relationship; Substrate Specificity; Sucrose; Topology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0077837

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.