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Escherichia coli K-12 substr. MG1655 Enzyme: GDP-fucose synthase



Gene: fcl Accession Numbers: EG11788 (EcoCyc), b2052, ECK2046

Synonyms: wcaG, yefB

Regulation Summary Diagram: ?

Subunit composition of GDP-fucose synthase = [Fcl]2

Summary:
GDP-fucose synthase is a bifunctional enzyme that catalyzes the two-step synthesis of GDP-fucose from GDP-4-dehydro-6-deoxy-D-mannose via a GDP-4-dehydro-6-L-deoxygalactose intermediate [Andrianopoulos98]. The presence of the GDP-4-dehydro-6-L-deoxygalactose intermediate was initially postulated, but not shown [Ginsburg61]. The epimerase reaction can occur in the absence of the NADPH cofactor [Menon99].

The enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family and the reductase-epimerase-dehydrogenase (RED) superfamily [Somers98, Rizzi98]. Its N-terminal domain contains a six-stranded NADP+ binding Rossmann fold domain [Rizzi98]. Crystal structure data suggests that there is a single active site [Somers98]. The reaction follows a random bi-bi mechanism [Menon99].

Mutants in proposed active site residues have been isolated and characterized [Rosano00].

The enzyme is a homodimer [Tonetti98, Rizzi98].

The enzymatic reaction has been shown to involve epimerization first at C-3'' and then at C-5'' of the mannose ring, followed by an NADPH-dependent reduction of the carbonyl at C-4''. The epimerization reactions involve the participation of acid/base residues at the active site [Lau08].

The enzyme as also been used in metabolic engineering studies to produce GDP-L-fucose for the synthesis of fucosylated oligosaccharides [Albermann01, Byun07].

Review: [Tonetti98a]

Locations: cytosol

Map Position: [2,124,249 <- 2,125,214] (45.78 centisomes)
Length: 966 bp / 321 aa

Molecular Weight of Polypeptide: 36.141 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0006801 , EchoBASE:EB1736 , EcoGene:EG11788 , EcoliWiki:b2052 , ModBase:P32055 , OU-Microarray:b2052 , PortEco:fcl , Protein Model Portal:P32055 , RefSeq:NP_416556 , RegulonDB:EG11788 , SMR:P32055 , String:511145.b2052 , UniProt:P32055

Relationship Links: InterPro:IN-FAMILY:IPR001509 , InterPro:IN-FAMILY:IPR016040 , PDB:Structure:1BSV , PDB:Structure:1BWS , PDB:Structure:1E6U , PDB:Structure:1E7Q , PDB:Structure:1E7R , PDB:Structure:1E7S , PDB:Structure:1FXS , PDB:Structure:1GFS , Pfam:IN-FAMILY:PF01370

In Paralogous Gene Group: 191 (7 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11]
GO:0009242 - colanic acid biosynthetic process Inferred by computational analysis [UniProtGOA12]
GO:0042351 - 'de novo' GDP-L-fucose biosynthetic process Inferred by computational analysis [UniProtGOA12, GOA06, GOA01]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0042803 - protein homodimerization activity Inferred from experiment [Rizzi98]
GO:0050577 - GDP-L-fucose synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Menon99]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11]
GO:0016853 - isomerase activity Inferred by computational analysis [UniProtGOA11]
GO:0050662 - coenzyme binding Inferred by computational analysis [GOA01]
GO:0070401 - NADP+ binding Inferred by computational analysis [GOA06]
Cellular Component: GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: cell processes adaptations dessication
cell structure capsule (M and K antigens)
cell structure surface antigens (ECA, O antigen of LPS)
metabolism biosynthesis of macromolecules (cellular constituents) colanic acid (M antigen)

Essentiality data for fcl knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Last-Curated ? 25-Jan-2013 by Fulcher C , SRI International


Enzymatic reaction of: GDP-fucose synthase

Synonyms: GMER, GFS, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, GDP-4-keto-6-deoxy-D-mannose epimerase/reductase

EC Number: 1.1.1.271

GDP-4-dehydro-α-D-rhamnose + NADPH + H+ <=> GDP-β-L-fucose + NADP+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown. [Lau08]

In Pathways: colanic acid building blocks biosynthesis , GDP-L-fucose biosynthesis I (from GDP-D-mannose)

Summary:
Studies have shown that the equilibrium of this reaction strongly favors production of GDP-L-fucose and NADP+ [Lau08].

Inhibitors (Competitive): GDP-L-fucose [Menon99] , NADP+ [Menon99] , GDP [Menon99]

Kinetic Parameters:

Substrate
Km (μM)
Citations
NADPH
9.0
[Menon99, BRENDA14]
GDP-4-dehydro-α-D-rhamnose
29.0
[Menon99]

pH(opt): 6 [Menon99]


Sequence Features

Feature Class Location Attached Group Citations Comment
Nucleotide-Phosphate-Binding-Region 10 -> 16 NADP+
[UniProt14]
UniProt: NADP.
Nucleotide-Phosphate-Binding-Region 36 -> 41 NADP+
[UniProt14]
UniProt: NADP.
Nucleotide-Phosphate-Binding-Region 105 -> 108 NADP+
[UniProt14]
UniProt: NADP.
Mutagenesis-Variant 107  
[Rosano00, UniProt13]
Alternate sequence: S → A; UniProt: Nearly abolishes catalytic activity. Minor effect of affinity for NADPH and substrate.
Amino-Acid-Site 107  
[UniProt13]
UniProt: Important for catalytic activity; Sequence Annotation Type: site.
Mutagenesis-Variant 109  
[UniProt13]
Alternate sequence: C → A; UniProt: Nearly abolishes catalytic activity.
Amino-Acid-Site 109  
[UniProt13]
UniProt: Important for catalytic activity; Sequence Annotation Type: site.
Mutagenesis-Variant 136  
[Rosano00, UniProt13]
Alternate sequence: Y → E; UniProt: Abolishes enzyme activity.
Active-Site 136  
[Rosano00, UniProt13]
UniProt: Proton donor/acceptor.
Mutagenesis-Variant 140  
[Rosano00, UniProt13]
Alternate sequence: K → S; UniProt: Nearly abolishes catalytic activity.
Alternate sequence: K → R; UniProt: Reduces catalytic activity 20-fold.
Amino-Acid-Sites-That-Bind 140  
[UniProt13]
UniProt: NADP.
Nucleotide-Phosphate-Binding-Region 163 -> 166 NADP+
[UniProt14]
UniProt: NADP.
Mutagenesis-Variant 179  
[Rosano00, UniProt13]
Alternate sequence: H → N; UniProt: Nearly abolishes catalytic activity.
Amino-Acid-Sites-That-Bind 179  
[UniProt13]
UniProt: NADP.
Mutagenesis-Variant 187  
[Rosano00, UniProt13]
Alternate sequence: R → A; UniProt: Decreases affinity for the substrate GDP-4-keto-6-deoxymannose.
Amino-Acid-Sites-That-Bind 187  
[UniProt13]
UniProt: Substrate; Non-Experimental Qualifier: probable.
Amino-Acid-Sites-That-Bind 202  
[UniProt13]
UniProt: Substrate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 209  
[UniProt13]
UniProt: Substrate; Non-Experimental Qualifier: by similarity.
Sequence-Conflict 255 -> 256  
[Aoyama94a, Stevenson96, UniProt10a]
Alternate sequence: EL → DV; UniProt: (in Ref. 1 and 2);
Amino-Acid-Sites-That-Bind 278  
[UniProt13]
UniProt: Substrate; Non-Experimental Qualifier: by similarity.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b2052 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11788; confirmed by SwissProt match.


References

Albermann01: Albermann C, Piepersberg W, Wehmeier UF (2001). "Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes." Carbohydr Res 334(2);97-103. PMID: 11502265

Andrianopoulos98: Andrianopoulos K, Wang L, Reeves PR (1998). "Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12." J Bacteriol 1998;180(4);998-1001. PMID: 9473059

Aoyama94a: Aoyama K, Haase AM, Reeves PR (1994). "Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli K-12." Mol Biol Evol 1994;11(6);829-38. PMID: 7815923

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Byun07: Byun SG, Kim MD, Lee WH, Lee KJ, Han NS, Seo JH (2007). "Production of GDP-L-fucose, L-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli." Appl Microbiol Biotechnol 74(4);768-75. PMID: 17111133

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

Ginsburg61: Ginsburg V (1961). "Studies on the biosynthesis of guanosine diphosphate L-fucose." J Biol Chem 236;2389-93. PMID: 13705522

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Lau08: Lau ST, Tanner ME (2008). "Mechanism and active site residues of GDP-fucose synthase." J Am Chem Soc 130(51);17593-602. PMID: 19053199

Menon99: Menon S, Stahl M, Kumar R, Xu GY, Sullivan F (1999). "Stereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli." J Biol Chem 274(38);26743-50. PMID: 10480878

Rizzi98: Rizzi M, Tonetti M, Vigevani P, Sturla L, Bisso A, Flora AD, Bordo D, Bolognesi M (1998). "GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily." Structure 6(11);1453-65. PMID: 9817848

Rosano00: Rosano C, Bisso A, Izzo G, Tonetti M, Sturla L, De Flora A, Bolognesi M (2000). "Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants." J Mol Biol 303(1);77-91. PMID: 11021971

Somers98: Somers WS, Stahl ML, Sullivan FX (1998). "GDP-fucose synthetase from Escherichia coli: structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site." Structure 6(12);1601-12. PMID: 9862812

Stevenson96: Stevenson G, Andrianopoulos K, Hobbs M, Reeves PR (1996). "Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid." J Bacteriol 1996;178(16);4885-93. PMID: 8759852

Tonetti98: Tonetti M, Rizzi M, Vigevani P, Sturla L, Bisso A, De Flora A, Bolognesi M (1998). "Preliminary crystallographic investigations of recombinant GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from E. coli." Acta Crystallogr D Biol Crystallogr 54 ( Pt 4);684-6. PMID: 9761875

Tonetti98a: Tonetti M, Sturla L, Bisso A, Zanardi D, Benatti U, De Flora A (1998). "The metabolism of 6-deoxyhexoses in bacterial and animal cells." Biochimie 80(11);923-31. PMID: 9893952

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

UniProt14: UniProt Consortium (2014). "UniProt version 2014-01 released on 2014-01-01 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Fri Nov 21, 2014, BIOCYC14B.