Kinetic Examination and Simulation of GDP-β-<scp>l</scp>-fucose Synthetase Reaction Using NADPH or NADH

Rentmeister, Andrea; Hoh, Christoph; Weidner, Stefan; Dräger, Gerald; Elling, Lothar; Liese, Andreas; Wandrey, Christian

doi:10.1080/10242420410001666362

Cited by 3 publications

(5 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the absence of any GER enzyme, but in the presence of any GMD, a broad peak of intermediate retention time was observed, as shown for Caenorhabditis GMD‐1a and Drosophila GMD (Fig. 4A,D); this presumably corresponds to the previously observed ketone and hydrate forms of GDP‐4‐keto‐6‐deoxymannose [40]. No intermediate product was formed in the absence of any GMD enzyme, and no GDP‐Fuc was formed in the absence of either GMD or GER, nor with the empty vector control, showing that the strain of E. coli used has no detectable GDP‐Fuc synthesis system.…”

Section: Resultssupporting

confidence: 74%

Reconstitution in vitro of the GDP‐fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

Rhomberg¹,

Fuchsluger²,

Rendić³

et al. 2006

The FEBS Journal

View full text Add to dashboard Cite

The deoxyhexose sugar fucose has an important fine‐tuning role in regulating the functions of glycoconjugates in disease and development in mammals. The two genetic model organisms Caenorhabditis elegans and Drosophila melanogaster also express a range of fucosylated glycans, and the nematode particularly has a number of novel forms. For the synthesis of such glycans, the formation of GDP‐fucose, which is generated from GDP‐mannose in three steps catalysed by two enzymes, is required. By homology we have identified and cloned cDNAs encoding these two proteins, GDP‐mannose dehydratase (GMD; EC 4.2.1.47) and GDP‐keto‐6‐deoxymannose 3,5‐epimerase/4‐reductase (GER or FX protein; EC 1.1.1.271), from both Caenorhabditis and Drosophila. Whereas the nematode has two genes encoding forms of GMD (gmd‐1 and gmd‐2) and one GER‐encoding gene (ger‐1), the insect has, like mammalian species, only one homologue of each (gmd and gmer). This compares to the presence of two forms of both enzymes in Arabidopsis thaliana. All corresponding cDNAs from Caenorhabditis and Drosophila, as well as the previously uncharacterized Arabidopsis GER2, were separately expressed, and the encoded proteins found to have the predicted activity. The biochemical characterization of these enzymes is complementary to strategies aimed at manipulating the expression of fucosylated glycans in these organisms.

show abstract

Section: Resultssupporting

confidence: 74%

Reconstitution in vitro of the GDP‐fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

Rhomberg¹,

Fuchsluger²,

Rendić³

et al. 2006

The FEBS Journal

View full text Add to dashboard Cite

show abstract

“…The substrate for GFS, GDP-6-deoxy-R-D-lyxo-hexos-4-ulose (GDP-6-deoxy-4-keto-mannose), was generated by treatment of GDP-mannose with GMD at room temperature. 15 Due to the sensitive nature of this compound toward decomposition, only minimal purification was employed, and the material was freshly prepared before each use. The reaction was monitored by mass spectral analysis until complete and the enzyme was removed by ultrafiltration.…”

Section: Resultsmentioning

confidence: 99%

“…This step introduces the characteristic 6-methyl group of fucose as well as the 4-keto functionality that is required for the subsequent epimerizations. The second step in the biosynthesis is catalyzed GDP-fucose synthase or GFS (also known as GDP-4-keto-6-deoxy- d -mannose epimerase/reductase or GMER). − GFS is remarkable in that it is able to catalyze three distinct reactions within a single active site. These include inversions of stereochemistry at both C-3′′ and C-5′′, as well as an NADPH-dependent reduction of the ketone functionality at C-4.…”

Section: Introductionmentioning

confidence: 99%

Mechanism and Active Site Residues of GDP-Fucose Synthase

Lau¹,

Tanner²

2008

J. Am. Chem. Soc.

View full text Add to dashboard Cite

L-fucose, 6-deoxy-L-galactose, is a key component of many important glycoconjugates including the blood group antigens and the Lewis(X) ligands. The biosynthesis of GDP-L-fucose begins with the action of a dehydratase that converts GDP-D-mannose into GDP-4-keto-6-deoxy-mannose. The enzyme GDP-fucose synthase, GFS, (also known as GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, GMER) then converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose. The GFS reaction involves epimerizations at both C-3'' and C-5'' followed by an NADPH-dependent reduction of the carbonyl at C-4. This manuscript describes studies that elucidate the order of the epimerization steps and the roles of the active site acid/base residues responsible for the epimerizations. An active site mutant, Cys109Ser, produces GDP-6-deoxy-D-altrose as its major product indicating that C-3'' epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. The same mutation results in the appearance of a kinetic isotope effect when [3''-(2)H]-GDP-6-deoxy-4-keto-mannose is used as a substrate. This indicates that Cys109 is the base responsible for the deprotonation of the substrate at C-3''. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5'' position of GDP-fucose in the presence of NADP(+). This confirms the order of epimerizations and the role of Cys109. Finally, the inactive His179Gln mutant readily catalyzes the wash-out of deuterium from the C-3'' position of [3''-(2)H]-GDP-6-deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3'' followed by C-5'') and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps.

show abstract

“…Then, mannose 6-phosphate is converted to GDP-4-keto-6-deoxymannose by GDP-mannose-4,6-dehydratase (Gmd, EC 4.2.1.47) and further to GDP- l -fucose by a bifunctional enzyme, GDP- l -fucose synthase (WcaG, EC 1.1.1.271), also named GDP-4-keto-6-deoxymannose 3,5-epimerase/4-reductase (Gmer), which generates the GDP-4-dehydro-6-deoxy- l -galactose intermediate by 3,5-epimerase activity and further generates GDP- l -fucose by 4-reductase activity. Among these five enzymes, ManC requires guanosine 5′-triphosphate (GTP) as the essential guanylyl donor, and WcaG for GDP- l -fucose synthesis requires NADPH or NADH as a cofactor …”

Section: Gdp-l-fucose Synthesismentioning

confidence: 99%

“…Among these five enzymes, ManC requires guanosine 5′-triphosphate (GTP) as the essential guanylyl donor, 15 and WcaG for GDP-L-fucose synthesis requires NADPH or NADH as a cofactor. 16 E. coli, the most frequently used host for 2′-FL biosynthesis, contains the complete endogenous pathway for de novo GDP-L-fucose synthesis. GDP-L-fucose plays an important role in biosynthesis of colanic acid, which is one of the main components of the E. coli cell wall.…”

Section: Gdp-l-fucose Synthesismentioning

confidence: 99%

Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

Liu

Zhu

Wang³

et al. 2022

J. Agric. Food Chem.

View full text Add to dashboard Cite

Human milk oligosaccharides (HMOs), a group of structurally diverse unconjugated glycans in breast milk, act as important prebiotics and have plenty of unique health effects for growing infants. 2′-Fucosyllactose (2′-FL) is the most abundant HMO, accounting for approximately 30%, among approximately 200 identified HMOs with different structures. 2′-FL can be enzymatically produced by α1,2-fucosyltransferase, using GDP-l-fucose as donor and lactose as acceptor. Metabolic engineering strategies have been widely used for enhancement of GDP-l-fucose supply and microbial production of 2′-FL with high productivity. GDP-l-fucose supply can be enhanced by two main pathways, including de novo and salvage pathways. 2′-FL-producing α1,2-fucosyltransferases have widely been identified from various microorganisms. Metabolic pathways for 2′-FL synthesis can be basically constructed by enhancing GDP-l-fucose supply and introducing α1,2-fucosyltransferase. Various strategies have been attempted to enhance 2′-FL production, such as acceptor enhancement, donor enhancement, and improvement of the functional expression of α1,2-fucosyltransferase. In this review, current progress in GDP-l-fucose synthesis and bacterial α1,2-fucosyltransferases is described in detail, various metabolic engineering strategies for enhancing 2′-FL production are comprehensively reviewed, and future research focuses in biotechnological production of 2′-FL are suggested.

show abstract

Kinetic Examination and Simulation of GDP-β-l-fucose Synthetase Reaction Using NADPH or NADH

Cited by 3 publications

References 37 publications

Reconstitution in vitro of the GDP‐fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

Reconstitution in vitro of the GDP‐fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

Mechanism and Active Site Residues of GDP-Fucose Synthase

Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

Contact Info

Product

Resources

About