Characterization of Helicobacter pylori α1,2‐Fucosyltransferase for Enzymatic Synthesis of Tumor‐Associated Antigens

Abstract

The a1,2-fucosyltransferase (a1,2-FucT) from Helicobacter pylori catalyzes the fucosylation of acceptor oligosaccharides at the C2-OH of terminal Galb units. The enzyme from strain NCTC11639 was evaluated for its ability to synthesize cancer-associated antigens. The a1,2-FucT was determined to be active over a pH range between 4.0 and 8.0 with the optimum occurring at pH 5.0. Although a divalent metal ion cofactor was not required for catalysis, enhancement of the enzyme activity was detected upon supplement with Mn 2+ . Detailed substrate specificity analysis revealed that a1,2-FucT can catalyze the fucosylation of a wide variety of oligosaccharide substrates. The a1,2-FucT preferentially fucosylated type 1 structure (Galb1-3GlcNAc)-containing glycans over type 2 structure (Galb1-4GlcNAc)-containing glycans. The Lewis a trisaccharide [Galb1-3-

Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation

“…In subsequent shake flask experiments, L-fucose and L-rhamnose (inductor of rha-promoter) were added 26 hour after induction of expression of recombinant genes, to allow sufficient time for the synthesis of LNT from lactose before onset of the fucosylation step. By doing so, the formation of large quantities of 2'-FL should be avoided, since lactose is a substrate for FutC, as well 9,22. Such an unfavorable byproduct formation had previously been described by Drouillard et al,11 where the in vivo synthesis of fucosylated LNnTled to the predominant synthesis of 2'-FL.…”

“…[15][16][17][18][19] Fucosylated derivatives of the core structure LNnT were synthesized in whole-cell cultivations, 10,11,20 while syntheses of fucosylated LNT structures so far have been only described from in vitro enzymatic conversions. [21][22][23] Enzymatic or whole-cell synthesis requires the availability of fucosyltransferases which can be used either as recombinant human genes, or from bacteria such as Helicobacter pylori. 23 Thus, fucosylation of LNT was carried out in vitro with H. pylori α1,2-or α1,3-fucosyltransferases, or with human α1,2-or α1,3/4-fucosyltransferases, whereas in vivo fucosylation reactions with LNnT were catalyzed by recombinant H. pylori fucosyltransferases in E. coli cells.…”

“…and 4 hours after induction at 37°C, cells were harvested by centrifugation (3,756 x g, 4°C for 15 min). Pellets were resuspended in HEPES buffer (100 mM, pH 7.6) and broken by sonication, followed by a second centrifugation step(22,410 x g, 4°C for 30 min). The resulting supernatants (cell-free extracts) were stored at -70°C until further use for in vitro experiments.In vitro experiments were conducted in HEPES buffer (100 mM, final conc., pH 7.6) with MnCl 2 (20 mM, final conc.…”

The one-pot multienzyme (OPME) synthesis of human blood group H antigens and a human milk oligosaccharide (HMOS) with highly active Thermosynechococcus elongatus α1–2-fucosyltransferase

“…Compared to GST-WbsJ, 11, 12 His 6 Prop-WgbL, 14 and Hp2FT, 7 His 6 -Te2FT showed a significantly higher k cat value for GDP-fucose (201-, 6.7-, and 14.6-fold, respectively) and a higher affinity for the acceptor. The catalytic efficiency of His 6 -Te2FT was similar to that of Hp2FT but is superior to WbsJ and WbgL as reflected by a higher k cat / K M v alue for GDP-Fuc (29.2-fold and 2.5-fold higher).…”

“…These include Helicobacter pylori ( H. pylori ) FutC (HpFutC or Hp2FT), 6, 7 Escherichia coli ( E. coli ) O86:B7 WbwK, 8 E. coli O86:K62:H2 WbnK, 9 E. coli O128 WbsJ, 10–12 and E. coli O127:K63(B8) WbiQ. 13 However, their expression levels are usually low which limit their application in synthesis.…”