EzCatDB: D00604
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DB codeD00604
RLCP classification8.131.1241100.8201 : Isomerization
8.113.1441400.8202 : Isomerization
9.5010.536200.8010 : Hydride transfer
CATH domainDomain 13.40.50.720 : Rossmann foldCatalytic domain
Domain 23.90.25.10 : UDP-galactose 4-epimerase; domain 1Catalytic domain
E.C.1.1.1.271
CSA1e7q
MACiEM0227

CATH domainRelated DB codes (homologues)
3.40.50.720 : Rossmann foldS00543,S00551,S00552,S00553,S00602,S00604,S00605,S00608,S00610,S00625,S00319,S00328,S00329,S00330,S00331,S00332,D00456,D00457,D00458,S00324,S00320,S00325,S00326,S00327,D00459,S00335,S00336,S00334,T00219,S00339,D00513,D00001,D00002,D00003,D00005,D00007,D00008,D00010,D00012,D00017,D00018,D00023,D00027,D00028,D00031,D00032,D00033,D00034,D00035,D00037,D00048,D00071,D00476,D00481,D00482,D00490,D00492,D00494,D00545,D00601,D00603,D00605,D00615,D00845,D00857,D00858,M00161,M00171,M00210,T00002,T00010,T00011,T00015,T00227,T00247,T00408,T00414,D00827,D00262,D00274,D00275,M00035,T00109
3.90.25.10 : UDP-galactose 4-epimerase; domain 1D00513,D00601,D00262,D00274,D00275

Enzyme Name
UniProtKBKEGG

P32055
Protein nameGDP-L-fucose synthetaseGDP-L-fucose synthase
GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase
SynonymsEC 1.1.1.271
GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase
RefSeqNP_416556.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_490294.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
PfamPF01370 (Epimerase)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00051Fructose and mannose metabolism
MAP00520Amino sugar and nucleotide sugar metabolism

UniProtKB:Accession NumberP32055
Entry nameFCL_ECOLI
ActivityGDP-L-fucose + NADP(+) = GDP-4-dehydro-6-deoxy-D-mannose + NADPH.
SubunitHomodimer.
Subcellular locationCytoplasm.
Cofactor

Compound table: links to PDB-related databases & PoSSuM

SubstratesProductsintermediates
KEGG-idC01222C00005C00080C00325C00006I00097I00098I00099I00100
CompoundGDP-4-dehydro-6-deoxy-D-mannoseNADPHH+GDP-L-fucoseNADP+GDP-6-deoxy-3,4-ene-mannoseGDP-6-deoxy-4-dehydro-altroseGDP-6-deoxy-4,5-ene-altroseGDP-6-deoxy-4-dehydro-L-galactose
Typeamide group,amine group,carbohydrate,nucleotideamide group,amine group,nucleotideothersamide group,amine group,carbohydrate,nucleotideamide group,amine group,nucleotide



ChEBI16955
16474
15378
13332
18009




PubChem439446
5884
1038
10918995
5886




                 
1bsvA01UnboundBound:NDP UnboundUnboundUnboundUnboundUnboundUnbound
1bwsA01UnboundBound:NDP UnboundUnboundUnboundUnboundUnboundUnbound
1e6uA01UnboundUnbound UnboundBound:NAPUnboundUnboundUnboundUnbound
1e7qA01UnboundUnbound UnboundBound:NAPUnboundUnboundUnboundUnbound
1e7rA01UnboundUnbound UnboundBound:NAPUnboundUnboundUnboundUnbound
1e7sA01UnboundUnbound UnboundBound:NAPUnboundUnboundUnboundUnbound
1fxsA01UnboundUnbound UnboundBound:NAPUnboundUnboundUnboundUnbound
1gfsA01UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1bsvA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1bwsA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1e6uA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1e7qA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1e7rA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1e7sA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1fxsA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound
1gfsA02UnboundUnbound UnboundUnboundUnboundUnboundUnboundUnbound

Active-site residues
resource
literature [5], [7], [9], [13], [14]
pdbCatalytic residuescomment
          
1bsvA01SER 107;CYS 109;TYR 136;LYS 140
 
1bwsA01SER 107;CYS 109;TYR 136;LYS 140
 
1e6uA01SER 107;CYS 109;TYR 136;LYS 140
 
1e7qA01       ;CYS 109;TYR 136;LYS 140
mutant S107A
1e7rA01SER 107;CYS 109;       ;LYS 140
mutant Y136E
1e7sA01SER 107;CYS 109;TYR 136;       
mutant K140R
1fxsA01SER 107;CYS 109;TYR 136;LYS 140
 
1gfsA01SER 107;CYS 109;TYR 136;LYS 140
 
1bsvA02HIS 179                        
 
1bwsA02HIS 179                        
 
1e6uA02HIS 179                        
 
1e7qA02HIS 179                        
 
1e7rA02HIS 179                        
 
1e7sA02HIS 179                        
 
1fxsA02HIS 179                        
 
1gfsA02HIS 179                        
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[5]Fig.7, p.1607-1608
[7]p.84-87
[9]p.1658-1659
[13]Scheme 12, p.9833-9834
[14]Fig.8, p.17598-17599

references
[1]
PubMed ID7742302
JournalBiochemistry
Year1995
Volume34
Pages6003-13
AuthorsJornvall H, Persson B, Krook M, Atrian S, Gonzalez-Duarte R, Jeffery J, Ghosh D
TitleShort-chain dehydrogenases/reductases (SDR).
[2]
CommentsX-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) IN COMPLEX WITH NADPH.
PubMed ID8805511
JournalStructure
Year1996
Volume4
Pages33-45
AuthorsTanaka N, Nonaka T, Nakanishi M, Deyashiki Y, Hara A, Mitsui Y
TitleCrystal structure of the ternary complex of mouse lung carbonyl reductase at 1.8 A resolution: the structural origin of coenzyme specificity in the short-chain dehydrogenase/reductase family.
Related PDB1cyd
Related UniProtKBP08074
[3]
CommentsX-RAY CRYSTALLOGRAPHY
PubMed ID9271498
JournalBiochemistry
Year1997
Volume36
Pages10675-84
AuthorsLiu Y, Thoden JB, Kim J, Berger E, Gulick AM, Ruzicka FJ, Holden HM, Frey PA
TitleMechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli.
Related PDB1kvu
[4]
CommentsX-RAY CRYSTALLOGRAPHY
PubMed ID9817848
JournalStructure
Year1998
Volume6
Pages1453-65
AuthorsRizzi M, Tonetti M, Vigevani P, Sturla L, Bisso A, Flora AD, Bordo D, Bolognesi M
TitleGDP-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.
Related PDB1bws
[5]
CommentsX-RAY CRYSTALLOGRAPHY (2.2 ANGSTROMS)
PubMed ID9862812
JournalStructure
Year1998
Volume6
Pages1601-12
AuthorsSomers WS, Stahl ML, Sullivan FX
TitleGDP-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.
Related PDB1bsv,1fxs,1gfs
Related UniProtKBP32055
[6]
PubMed ID10480878
JournalJ Biol Chem
Year1999
Volume274
Pages26743-50
AuthorsMenon S, Stahl M, Kumar R, Xu GY, Sullivan F
TitleStereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli.
[7]
CommentsX-RAY CRYSTALLOGRAPHY
PubMed ID11021971
JournalJ Mol Biol
Year2000
Volume303
Pages77-91
AuthorsRosano C, Bisso A, Izzo G, Tonetti M, Sturla L, De Flora A, Bolognesi M
TitleProbing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.
Related PDB1e6u,1e7q,1e7r,1e7s
[8]
PubMed ID10896473
JournalStructure
Year2000
Volume8
Pages453-62
AuthorsDeacon AM, Ni YS, Coleman WG Jr, Ealick SE
TitleThe crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist.
Related PDB1eq2
Related UniProtKBP67910
[9]
PubMed ID11706991
JournalCell Mol Life Sci
Year2001
Volume58
Pages1650-65
AuthorsAllard ST, Giraud MF, Naismith JH
TitleEpimerases: structure, function and mechanism.
[10]
PubMed ID15023057
JournalBiochemistry
Year2004
Volume43
Pages3057-67
AuthorsVogan EM, Bellamacina C, He X, Liu HW, Ringe D, Petsko GA
TitleCrystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis.
[11]
PubMed ID15823050
JournalBiochemistry
Year2005
Volume44
Pages5907-15
AuthorsMorrison JP, Read JA, Coleman WG Jr, Tanner ME
TitleDismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism.
[12]
CommentsX-RAY CRYSTALLOGRAPHY (1.4 ANGSTROMS) OF 1-377 IN COMPLEX WITH NAD AND SUBSTRATES, SUBUNIT, MUTAGENESIS OF CYS-145; TYR-174; LYS-178; LYS-217 AND ARG-306.
PubMed ID16366586
JournalJ Am Chem Soc
Year2005
Volume127
Pages18309-20
AuthorsMajor LL, Wolucka BA, Naismith JH
TitleStructure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.
Related PDB2c5A,2c5e,2c54,2c59
Related UniProtKBQ93VR3
[13]
PubMed ID19058170
JournalAngew Chem Int Ed Engl
Year2008
Volume47
Pages9814-59
AuthorsThibodeaux CJ, Melancon CE 3rd, Liu HW
TitleNatural-product sugar biosynthesis and enzymatic glycodiversification.
[14]
PubMed ID19053199
JournalJ Am Chem Soc
Year2008
Volume130
Pages17593-602
AuthorsLau ST, Tanner ME
TitleMechanism and active site residues of GDP-fucose synthase.

comments
This enzyme is a distant homologue of the short-chain dehydrogenase/reductase (SDR) superfamily, which includes Drosophia alcohol dehydrogenase (S00319 in EzCatDB). This enzyme has got a catalytic triad composed of conserved residues, Ser, Tyr, and Lys. This enzyme is more closely related to dTDP-glucose 4,6-dehydratase (EC 4.2.1.46; D00262 in EzCatDB) and UDP-glucose 4-epimerase (EC 5.1.3.2; D00274 in EzCatDB).
This enzyme catalyzes three reactions, two epimerizations and a reduction. According to the literature [6] and [7], although this enzyme can catalyze the epimerization reaction without NADP or NADPH, these coenzymes can assist the reaction. According to the literature, Tyr136 and Lys140 seems to be involved in the epimerization, along with Cys109 and His179. As in the reduction reaction by the SDR family, Lys140 may modulate the activity of Tyr136 through the 2'-OH of NADP(H).
According to the literature [14], Cys109 acts as a general base to deprotonate C3' atom of the substrate in the first epimerization, and C5' atom of the intermediate in the second epimerization, whereas His179 serves as a general acid to protonate the C3' atom from the opposite side in the first epimerization, and the the C5' atom in the second epimerization. For the same residues to play the role as either general base or acid in the two consecutive reaction steps, there must be a proton shuttle mechanism to and from the active site during the lifetime of the GDP-6-deoxy-4-dehydro-altrose intermediate, in order to recover the protonation state of these catalytic residues. However, such proton shuttle system has not be found so far. (Ser107/Tyr136 can be involved in the proton shuttle, though.)
Taken together, this enzyme catalyze the following reactions.
(A) Isomerization from GDP-4-dehydro-6-deoxy-D-mannose to GDP-6-deoxy-3,4-ene-mannose (I00097):
(A0) Lys140 modulates the activity (or pKa) of Tyr136 via 2'-hydroxyl group of NADPH.
(A1) Cys109 acts as a general base to deprotonate C3' atom of the substrate, leading to the negative charge formation at the carbonyl oxygen at C4' position, to produce the intermediate (I00097). Here, Tyr136 may stabilize the negative charge on the O4'.
(B) Isomerization from GDP-6-deoxy-3,4-ene-mannose to GDP-6-deoxy-4-dehydro-altrose (I00098):
(B0) Lys140 modulates the activity (or pKa) of Tyr136 via 2'-hydroxyl group of NADPH. Tyr136 may stabilize the negative charge on the O4'.
(B1) His179 acts as a general acid to protonate the C3' atom of the intermediate (I00097). Simultaneously, the negative charge on the O4 decreases, leading to the formation of the second intermediate (I00098).
##Here, the protonation states of Cys109 and His179 must be recovered somehow.
(C) Isomerization from GDP-6-deoxy-4-dehydro-altrose to GDP-6-deoxy-4,5-ene-altrose (I00099):
(C0) Lys140 modulates the activity (or pKa) of Tyr136 via 2'-hydroxyl group of NADPH.
(C1) Cys109 acts as a general base to deprotonate C5' atom of the second intermediate (I00098), leading to the negative charge formation at the carbonyl oxygen at C4' position, to produce the third intermediate (I00099). Here, Tyr136 may stabilize the negative charge on the O4'.
(D) Isomerization from GDP-6-deoxy-4,5-ene-altrose to GDP-6-deoxy-4-dehydro-L-galactose (I00100):
(D0) Lys140 modulates the activity (or pKa) of Tyr136 via 2'-hydroxyl group of NADPH. Tyr136 may stabilize the negative charge on the O4'.
(D1) His179 acts as a general acid to protonate the C5' atom of the third intermediate (I00099). Simultaneously, the negative charge on the O4 decreases, leading to the formation of the fourth intermediate (I00100).
(E) Hydride transfer from NADPH to C5' atom of GDP-6-deoxy-4-dehydro-L-galactose:
(E0) Lys140 modulates the activity (or pKa) of Tyr136 via 2'-hydroxyl group of NADPH, along with the N1 atom of the nicotinamide group in NADPH, whereas Ser107 modulates the pKa of carbonyl oxygen of the intermediate (I00100).
(E1) Tyr136 acts as a general acid to protonate the carbonyl oxygen of the substrate. Meanwhile, the hydride transfer occurs from the C4 atom of the nicotinamide to the carbonyl carbon of the intermediate (I00100).

createdupdated
2010-08-062011-08-10


Copyright: Nozomi Nagano, JST & CBRC-AIST
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Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
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Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
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