title: Engineering the product profile of a polysialyltransferase.
abstract: Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Keys
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Hazel L S
authors.initials: HL
authors.lastname: Fuchs
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Ehrit
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Alves
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Freiberger
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D040961:Genetic Drift; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D038541:Neisseria meningitidis, Serogroup B / Q000201:enzymology*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis / Q000737:chemistry; D015202:Protein Engineering; D012794:Sialic Acids / Q000737:chemistry; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics*
chemical_list: D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; C021319:polysialic acid; D012799:Sialyltransferases
references.citation: Chembiochem. 2013 Oct 11;14(15):1949-53
references.pmid: 24009103
references.citation: J Bacteriol. 2011 Apr;193(7):1576-82
references.pmid: 21278299
references.citation: Nat Rev Mol Cell Biol. 2009 Dec;10(12):866-76
references.pmid: 19935669
references.citation: Glycobiology. 1992 Feb;2(1):5-23
references.pmid: 1550990
references.citation: J Biol Chem. 2012 Nov 2;287(45):38135-49
references.pmid: 22989876
references.citation: Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20753-8
references.pmid: 24302764
references.citation: Glycobiology. 2013 Jul;23(7):764-77
references.pmid: 23481097
references.citation: PLoS One. 2013 Jul 26;8(7):e69888
references.pmid: 23922842
references.citation: J Am Chem Soc. 2011 Aug 17;133(32):12758-66
references.pmid: 21739979
references.citation: Nat Rev Neurosci. 2008 Jan;9(1):26-35
references.pmid: 18059411
references.citation: Annu Rev Microbiol. 2011;65:563-81
references.pmid: 21721938
references.citation: J Biol Chem. 2007 Dec 21;282(51):36777-81
references.pmid: 17981795
references.citation: Anal Chem. 2007 Sep 15;79(18):7161-9
references.pmid: 17705556
references.citation: BMC Biol. 2007 Jul 17;5:29
references.pmid: 17640347
references.citation: Anal Biochem. 2012 Aug 1;427(1):60-8
references.pmid: 22579847
references.citation: J Biol Chem. 2012 Mar 2;287(10):7203-12
references.pmid: 22235128
references.citation: Protein Sci. 2001 Sep;10(9):1699-711
references.pmid: 11514661
references.citation: Nat Methods. 2006 Aug;3(8):609-14
references.pmid: 16862135
references.citation: Cell. 2006 Sep 8;126(5):847-50
references.pmid: 16959564
references.citation: Proc Natl Acad Sci U S A. 2009 Jul 21;106(29):11851-6
references.pmid: 19571009
references.citation: Mol Microbiol. 2007 Sep;65(5):1258-75
references.pmid: 17662040
references.citation: Nat Protoc. 2007;2(4):924-32
references.pmid: 17446874
references.citation: Ann N Y Acad Sci. 1994 Jul 29;726:118-31
references.pmid: 8092670
references.citation: PLoS One. 2009 Sep 14;4(9):e7002
references.pmid: 19759823
references.citation: Adv Exp Med Biol. 2010;663:137-47
references.pmid: 20017020
references.citation: Annu Rev Biochem. 2006;75:39-68
references.pmid: 16756484
references.citation: J Mol Biol. 2008 Jun 20;379(5):1029-44
references.pmid: 18495157
references.citation: Glycobiology. 2007 Jul;17(7):735-43
references.pmid: 17384120
references.citation: Biotechnol Bioeng. 2007 Oct 15;98(3):599-610
references.pmid: 17394253
references.citation: Glycobiology. 2014 Feb;24(2):150-8
references.pmid: 24259400
references.citation: Glia. 2012 May;60(6):979-92
references.pmid: 22460918
references.citation: J Biol Chem. 2004 Oct 1;279(40):42345-9
references.pmid: 15299014
references.citation: J Biol Chem. 2012 Sep 21;287(39):32770-9
references.pmid: 22851175
references.citation: J Biol Chem. 2003 Apr 25;278(17):15349-59
references.pmid: 12578835
references.citation: Bioconjug Chem. 2008 Mar;19(3):643-50
references.pmid: 18307285
references.citation: Anal Biochem. 2012 Aug 15;427(2):107-15
references.pmid: 22617798
references.citation: Nat Methods. 2008 Nov;5(11):939-42
references.pmid: 18931667
references.citation: Proc Natl Acad Sci U S A. 2011 May 3;108(18):7397-402
references.pmid: 21502532
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 7;103(45):16989-94
references.pmid: 17075041
references.citation: Glycoconj J. 2013 Dec;30(9):857-70
references.pmid: 23949787
references.citation: Proc Natl Acad Sci U S A. 2009 Jul 21;106(29):11995-2000
references.pmid: 19587240
references.citation: Nat Rev Immunol. 2006 Nov;6(11):849-58
references.pmid: 17024229
references.citation: Nat Rev Immunol. 2009 Mar;9(3):213-20
references.pmid: 19214194
references.citation: Annu Rev Biochem. 2005;74:433-80
references.pmid: 15952894
references.citation: Adv Biochem Eng Biotechnol. 2004;90:89-111
references.pmid: 15453186
references.citation: Top Curr Chem. 2015;366:55-96
references.pmid: 23715636
references.citation: Glycobiology. 2001 Sep;11(9):759-67
references.pmid: 11555620
references.citation: Mol Microbiol. 2008 Jun;68(5):1252-67
references.pmid: 18435708