Targeted Fucosylation of Glycans with Engineered Bacterial Fucosyltransferase Variants

Abstract

Fucosyltransferases (FucTs) are crucial for the synthesis of Lewis-type glycan epitopes. The synthetic capacity of efficient bacterial enzymes and their variants has not yet been fully exploited. In the present work, we investigated two previously described variants of α1,3FucT from Helicobacter pylori strains for their flexibility in substrate utilization and their applicability in the enzymatic synthesis of Lewis epitopes. We used the truncated enzyme variant of FutA from H. pylori 26695 (FucTΔ52A128N/H129E/Y132I/S46F, FucTΔ52-4M) and the trun-cated α3FucT from H. pylori NCTC11639 (FucTΔ66). N-Acetyllactosamine type 1 and type 2 as well as N',N''-diacetyllactosamine were investigated as substrates. Both FucT variants exhibit α1,3/ 4FucT activity. Novel glycan structures were obtained displaying Lewis blood group antigens in a site-specific sequence. Fucosylated N',N''-diacetyllactosamine was synthesized for the first time with FucTΔ52-4M. Our work paves the way for targeted fucosylation patterns that can be tested for lectin binding.

Exploring Regioselective Fucosylation Catalyzed by Bacterial Glycosyltransferases through Substrate Promiscuity and Acceptor-Mediated Glycosylation

“…Unfortunately, the substrate specificity of bacterial enzymes is more flexible, especially enzymes involved in the fucosylation or sialylation of glycan chains with similar structures, and heterogeneous glycans are produced. Performing detailed studies on the substrate selectivities of enzymes is a common approach to produce the expected products; however, this process is usually accompanied by a small amount of undesired regioisomers. − Although a mutated enzyme was successfully demonstrated to enhance the catalytic activity and substrate specificities in fucosylation reactions, , the mutation study was time-consuming and only provided slightly favorable results. In contrast, using mammalian glycoenzymes can efficiently prevent these promiscuities against acceptors; however, these post-translationally modified enzymes are expensive and not easy to access. , Recently, acceptor engineering strategies have been developed for site-specific fucosylation by creating steric hindrance in retrofitted substrates.…”

“…Performing detailed studies on the substrate selectivities of enzymes is a common approach to produce the expected products; however, this process is usually accompanied by a small amount of undesired regioisomers. 25−27 Although a mutated enzyme was successfully demonstrated to enhance the catalytic activity and substrate specificities in fucosylation reactions, 28,29 the mutation study was time-consuming and only provided slightly favorable results. In contrast, using mammalian glycoenzymes can efficiently prevent these promiscuities against acceptors; however, these post-translationally modified enzymes are expensive and not easy to access.…”