title: Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.
abstract: The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts.
authors: Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, CA, 95616, USA.
authors: Yanhong
authors: Y
authors: Li
authors: Hai
authors: H
authors: Yu
authors: Vireak
authors: V
authors: Thon
authors: Yi
authors: Y
authors: Chen
authors: Musleh M
authors: MM
authors: Muthana
authors: Jingyao
authors: J
authors: Qu
authors: Liana
authors: L
authors: Hie
authors: Xi
authors: X
authors: Chen
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D016979:Pasteurella multocida / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D016695:Glycosyltransferases; C413756:KfiA protein, E coli; D017351:N-Acetylglucosaminyltransferases; C450833:heparosan synthase; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase
references: J Biol Chem. 2010 Jan 15;285(3):1597-606
references: 19915003
references: J Biol Chem. 2010 Oct 29;285(44):34240-9
references: 20729556
references: Biochem J. 1997 Sep 15;326 ( Pt 3):929-39
references: 9334165
references: Carbohydr Res. 2002 Sep 27;337(17):1547-52
references: 12350324
references: J Biol Chem. 2012 Mar 2;287(10):7203-12
references: 22235128
references: Vet Rec. 1994 Feb 19;134(8):191-2
references: 8171794
references: Vet Microbiol. 2010 Sep 28;145(1-2):90-9
references: 20362405
references: J Biol Chem. 2000 Sep 1;275(35):27311-5
references: 10859322
references: Glycoconj J. 2000 Jul-Sep;17(7-9):597-605
references: 11421351
references: J Biol Chem. 2002 Mar 1;277(9):7209-13
references: 11756462
references: Chem Commun (Camb). 2011 Oct 14;47(38):10815-7
references: 21863157
references: Org Biomol Chem. 2007 Mar 21;5(6):865-72
references: 17340000
references: IUBMB Life. 2002 Oct;54(4):163-75
references: 12512855
references: J Clin Invest. 2001 Jul;108(2):169-73
references: 11457867
references: Appl Microbiol Biotechnol. 2007 Feb;74(2):263-72
references: 17131147
references: Adv Carbohydr Chem Biochem. 2012;67:95-136
references: 22794183
references: Appl Microbiol Biotechnol. 2010 Feb;85(6):1881-91
references: 19756580
references: J Bacteriol. 2004 Dec;186(24):8529-32
references: 15576804
references: J Biol Chem. 2006 Jan 27;281(4):1929-34
references: 16303756
references: Biochim Biophys Acta. 2002 Dec 19;1573(3):346-55
references: 12417417
references: Thromb Haemost. 1991 Jul 12;66(1):44-8
references: 1926051
references: J Biol Chem. 2011 Jan 21;286(3):1777-85
references: 21084307
references: J Mol Biol. 2003 Apr 25;328(2):307-17
references: 12691742
references: Science. 2011 Oct 28;334(6055):498-501
references: 22034431
references: Nature. 1998 Jul 2;394(6688):85-8
references: 9665133
references: Glycobiology. 2002 Jan;12(1):9R-16R
references: 11825882
references: Chem Commun (Camb). 2012 Mar 11;48(21):2728-30
references: 22306833
references: Biochemistry. 2006 Oct 10;45(40):12358-65
references: 17014088
references: Eur J Biochem. 1981 May 15;116(2):359-64
references: 7018909
references: Appl Microbiol Biotechnol. 2012 Sep;95(5):1199-210
references: 22198719
references: J Biol Chem. 2007 Sep 28;282(39):28321-28327
references: 17627940
references: Anat Rec. 2002 Nov 1;268(3):317-26
references: 12382327