Characterization of WbiQ: An α1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen.
Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an α1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-β1,3-GalNAc-α-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe, on a milligram scale.
Pettit
Nicholas
N
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
Styslinger
Thomas
T
Mei
Zhen
Z
Han
Weiqing
W
Zhao
Guohui
G
Wang
Peng George
PG
D001789:Blood Group Antigens / Q000096:biosynthesis*; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology*; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019081:O Antigens / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D001789:Blood Group Antigens; D005647:Fucosyltransferases; D019081:O Antigens; D011993:Recombinant Fusion Proteins; D000097631:Galactoside 2-alpha-L-fucosyltransferase
Glycobiology. 2003 Jul;13(7):41R-53R
12651883
Mol Microbiol. 1999 Feb;31(4):1265-74
10096092
J Biol Chem. 2007 Dec 14;282(50):36593-602
17913713
J Bacteriol. 2001 Dec;183(24):7067-75
11717264
Biochem J. 2005 Jul 1;389(Pt 1):63-72
15766331
Carbohydr Res. 2008 Aug 11;343(12):1952-65
18279843
J Immunogenet. 1990 Aug-Oct;17(4-5):279-99
2093727
Carbohydr Res. 1993 Sep 2;247:255-62
7693348
J Biol Chem. 1995 Apr 14;270(15):8844-50
7721792
J Bacteriol. 2001 Aug;183(16):4779-85
11466281
Glycobiology. 2003 Jun;13(6):445-55
12626397
Gut. 1991 Mar;32(3):287-93
1826491
Microbiology (Reading). 1999 Nov;145 ( Pt 11):3245-3253
10589734
Glycobiology. 2008 Apr;18(4):290-302
18252778
Biochemistry. 2008 Nov 4;47(44):11590-7
18842005
FEMS Microbiol Rev. 2002 Mar;26(1):17-47
12007641
Biochemistry. 2008 Feb 5;47(5):1241-8
18179256
Glycobiology. 2007 May;17(5):455-66
17172260
FEMS Microbiol Rev. 2006 May;30(3):382-403
16594963
J Am Chem Soc. 2005 Feb 23;127(7):2040-1
15713070
Annu Rev Biochem. 2008;77:521-55
18518825
J Biol Chem. 1992 Feb 5;267(4):2737-44
1733969
Nat Protoc. 2010 Apr;5(4):636-46
20224564
Proc Natl Acad Sci U S A. 1990 Sep;87(17):6674-8
2118655
Medicina (B Aires). 2000;60(2):249-52
10962819
J Immunogenet. 1990 Aug-Oct;17(4-5):235-45
2093726
Glycobiology. 2006 Dec;16(12):158R-184R
16973733
Biochemistry. 2008 Jan 8;47(1):378-87
18078329