Kinetic Dissection of the Reaction of Human GDP-l-Fucose Synthase.

Smyshliaev, D; Pfeiffer, M; Oppermann, U; Nidetzky, B

Journal article

Kinetic Dissection of the Reaction of Human GDP-l-Fucose Synthase.

Abstract:: GDP-l-fucose is a universal sugar donor for the cellular biosynthesis of l-fucose-containing glycans. Its supply comes primarily from the reaction of GDP-l-fucose synthase (GFS), also known as GDP-4″-keto-6″-deoxy-d-mannose epimerase/reductase. GFS converts GDP-4″-keto-6″-deoxy-d-mannose by epimerization at both C-3″ and C-5″ followed by NADPH-dependent reduction of the carbonyl at C-4″. Here, we report kinetic and structural characterization of human GFS with the aim of dissecting the multistep pathway of the enzymatic reaction. Kinetic isotope effects due to [3″-2H] or [5″-2H] in GDP-4″-keto-6″-deoxy-d-mannose and [4S-2H] in NADPH were masked in the steady-state rate of the wild-type enzyme, indicating that the immediate catalytic steps were not rate-limiting for the overall reaction. An isotope effect, however, appeared with GFS variants defective in catalysis of an elementary step, when the isotope probe for that particular step was used in the reaction (C116S: C-3″ epimerization, [3″-2H]-substrate; Y143F: 4″-keto group reduction, [4S-2H]-NADPH). Evidence from steady-state and transient kinetic studies combined with reaction simulations revealed that GFS uses a random mechanism of substrate binding and product release, with the peculiarity that at saturating conditions of substrate and NADPH, the product dissociation happens from abortive GFS complexes with NADPH/GDP-l-fucose and NADP+/GDP-4″-keto-6″-deoxy-d-mannose and involves GDP-l-fucose release as the rate-determining step. GFS complex structures with NADP+ and NADP+/GDP suggest an induced-fit conformational change required to unbind the GDP moiety for dissociation from the enzyme as the molecular cause of the slow product release. Collectively, these results establish the basic kinetic framework of the GFS reaction, which is critical for understanding this important enzyme mechanistically and in its role as an inhibitor target to control glycan fucosylation in vivo.

Publication status:: Published

Peer review status:: Peer reviewed

Actions

Email

Email this record

Send the bibliographic details of this record to your email address.

Your Email
Please enter the email address that the record information will be sent to.

-
Your message (optional)
Please add any additional information to be included within the email.
Cite

Cite this record

APA Style

Smyshliaev, D., Pfeiffer, M., Oppermann, U., & Nidetzky, B. (2025). Kinetic Dissection of the Reaction of Human GDP-l-Fucose Synthase. . ACS Catalysis, 15(16), 13872–13885.

MLA Style

Smyshliaev, D., et al. “Kinetic Dissection of the Reaction of Human GDP-l-Fucose Synthase. .” ACS Catalysis, vol. 15, no. 16, American Chemical Society, 2025, pp. 13872–85.

Chicago Style

Smyshliaev, D, M Pfeiffer, U Oppermann, and B Nidetzky. 2025. “Kinetic Dissection of the Reaction of Human GDP-l-Fucose Synthase. .” ACS Catalysis 15 (16): 13872–85.
Share
Print