title: Isolation of mutant alginate lyases with cleavage specificity for di-guluronic acid linkages.
abstract: Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1-4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca(2+), one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II' suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.
authors.affiliation: Department of Biotechnology, SINTEF Materials and Chemistry, N-7465 Trondheim, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Tøndervik
authors.forename: Geir
authors.initials: G
authors.lastname: Klinkenberg
authors.forename: Olav A
authors.initials: OA
authors.lastname: Aarstad
authors.forename: Finn
authors.initials: F
authors.lastname: Drabløs
authors.forename: Helga
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: Trond E
authors.initials: TE
authors.lastname: Ellingsen
authors.forename: Gudmund
authors.initials: G
authors.lastname: Skjåk-Bræk
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
authors.forename: Håvard
authors.initials: H
authors.lastname: Sletta
mesh_terms: D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D015723:Gene Library; D006603:Hexuronic Acids / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D009844:Oligosaccharides; C007896:guluronic acid; C008324:mannuronic acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase
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