Isolation of mutant alginate lyases with cleavage specificity for di-guluronic acid linkages.
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.
Tøndervik
Anne
A
Department of Biotechnology, SINTEF Materials and Chemistry, N-7465 Trondheim, Norway.
Klinkenberg
Geir
G
Aarstad
Olav A
OA
Drabløs
Finn
F
Ertesvåg
Helga
H
Ellingsen
Trond E
TE
Skjåk-Bræk
Gudmund
G
Valla
Svein
S
Sletta
Håvard
H
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
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
FEBS Lett. 2009 Apr 17;583(8):1323-6
19306878
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
Acta Chem Scand. 1967;21(1):98-110
6031334
Nucleic Acids Res. 2000 Jan 1;28(1):235-42
10592235
Biotechnol Lett. 2006 Nov;28(21):1701-12
16912921
Carbohydr Res. 1990 Apr 2;198(1):101-9
2191774
J Protein Chem. 1998 Jan;17(1):29-36
9491925
Curr Diab Rep. 2007 Aug;7(4):314-20
17686410
J Biol Chem. 2004 Sep 10;279(37):38749-54
15247257
Dan Med Bull. 2008 Feb;55(1):72-7
18321446
Appl Environ Microbiol. 2007 Feb;73(3):906-12
17142370
J Biol Chem. 2004 Jul 23;279(30):31863-72
15136569
Carbohydr Res. 1971 Apr;17(2):297-308
5150892
J Chromatogr A. 2004 Feb 13;1026(1-2):271-81
14763754
Carbohydr Res. 1998 Jun;308(3-4):409-15
11001631
Bioinformatics. 1999 Apr;15(4):327-32
10320401
Can J Microbiol. 1974 Jul;20(7):993-8
4858117
Biomacromolecules. 2006 Jul;7(7):2108-21
16827577
J Chromatogr A. 2005 Nov 4;1093(1-2):59-68
16233871
J Gen Microbiol. 1981 Jul;125(1):217-20
6801192
J Mol Biol. 2005 Sep 9;352(1):11-21
16081095
Appl Environ Microbiol. 2009 Apr;75(7):2002-11
19201973
J Mater Sci Mater Med. 2005 Jun;16(6):503-9
15928864
Carbohydr Res. 1977 Aug;57:163-71
332364
Carbohydr Res. 1986 Oct 15;154:239-50
3098421
Gene. 1994 May 27;143(1):61-6
8200539
J Pharm Sci. 2008 Aug;97(8):2892-923
17963217
Biochim Biophys Acta. 1969 Dec 30;192(3):557-9
5368261
Protein Eng. 1995 Feb;8(2):127-34
7630882
Nature. 1964 Oct 10;204:187-8
14222269
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
9254694
Curr Pharm Des. 2008;14(29):3168-95
19075698
J Wound Care. 2000 Feb;9(2):56-60
11933281
J Mol Biol. 2008 Jul 4;380(2):373-85
18514736
Annu Rev Microbiol. 2000;54:289-340
11018131
J Mol Biol. 2005 Feb 4;345(5):1111-8
15644208
J Control Release. 2007 May 14;119(1):5-24
17382422
Mol Microbiol. 1995 May;16(4):719-31
7476166
J Bacteriol. 2004 May;186(9):2891-6
15090531