DB code: D00604

RLCP classification	8.131.1241100.8201 : Isomerization
	8.113.1441400.8202 : Isomerization
	9.5010.536200.8010 : Hydride transfer
CATH domain	3.40.50.720 : Rossmann fold	Catalytic domain
CATH domain	3.90.25.10 : UDP-galactose 4-epimerase; domain 1	Catalytic domain
E.C.	1.1.1.271
CSA	1e7q
M-CSA	1e7q
MACiE	M0227

CATH domain	Related DB codes (homologues)
3.40.50.720 : Rossmann fold	S00543 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 1	D00513 D00601 D00262 D00274 D00275

CATH domain

Related DB codes (homologues)

3.40.50.720 : Rossmann fold

S00543 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 1

D00513 D00601 D00262 D00274 D00275

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	RefSeq	Pfam
P32055	GDP-L-fucose synthetase	EC 1.1.1.271 GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase	NP_416556.1 (Protein) NC_000913.2 (DNA/RNA sequence) YP_490294.1 (Protein) NC_007779.1 (DNA/RNA sequence)	PF01370 (Epimerase) [Graphical View]

KEGG enzyme name
GDP-L-fucose synthase GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P32055	FCL_ECOLI	GDP-L-fucose + NADP(+) = GDP-4-dehydro-6-deoxy-D-mannose + NADPH.	Homodimer.	Cytoplasm.

KEGG Pathways
Map code	Pathways	E.C.
MAP00051	Fructose and mannose metabolism
MAP00520	Amino sugar and nucleotide sugar metabolism

Compound table
	Substrates			Products		Intermediates
KEGG-id	C01222	C00005	C00080	C00325	C00006	I00097	I00098	I00099	I00100
E.C.
Compound	GDP-4-dehydro-6-deoxy-D-mannose	NADPH	H+	GDP-L-fucose	NADP+	GDP-6-deoxy-3,4-ene-mannose	GDP-6-deoxy-4-dehydro-altrose	GDP-6-deoxy-4,5-ene-altrose	GDP-6-deoxy-4-dehydro-L-galactose
Type	amide group,amine group,carbohydrate,nucleotide	amide group,amine group,nucleotide	others	amide group,amine group,carbohydrate,nucleotide	amide group,amine group,nucleotide
ChEBI	16955 16955	16474 16474	15378 15378	13332 13332	18009 18009
PubChem	439446 439446	5884 5884	1038 1038	10918995 10918995	5886 5886

1bsvA01	Unbound	Bound:NDP		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1bwsA01	Unbound	Bound:NDP		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1e6uA01	Unbound	Unbound		Unbound	Bound:NAP	Unbound	Unbound	Unbound	Unbound
1e7qA01	Unbound	Unbound		Unbound	Bound:NAP	Unbound	Unbound	Unbound	Unbound
1e7rA01	Unbound	Unbound		Unbound	Bound:NAP	Unbound	Unbound	Unbound	Unbound
1e7sA01	Unbound	Unbound		Unbound	Bound:NAP	Unbound	Unbound	Unbound	Unbound
1fxsA01	Unbound	Unbound		Unbound	Bound:NAP	Unbound	Unbound	Unbound	Unbound
1gfsA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1bsvA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1bwsA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1e6uA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1e7qA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1e7rA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1e7sA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1fxsA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1gfsA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [5], [7], [9], [13], [14]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1bsvA01	SER 107;CYS 109;TYR 136;LYS 140
1bwsA01	SER 107;CYS 109;TYR 136;LYS 140
1e6uA01	SER 107;CYS 109;TYR 136;LYS 140
1e7qA01	;CYS 109;TYR 136;LYS 140				mutant S107A
1e7rA01	SER 107;CYS 109;;LYS 140				mutant Y136E
1e7sA01	SER 107;CYS 109;TYR 136;				mutant K140R
1fxsA01	SER 107;CYS 109;TYR 136;LYS 140
1gfsA01	SER 107;CYS 109;TYR 136;LYS 140
1bsvA02	HIS 179
1bwsA02	HIS 179
1e6uA02	HIS 179
1e7qA02	HIS 179
1e7rA02	HIS 179
1e7sA02	HIS 179
1fxsA02	HIS 179
1gfsA02	HIS 179

References for Catalytic Mechanism
References	Sections	No. 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]
Resource
Comments
Medline ID
PubMed ID	7742302
Journal	Biochemistry
Year	1995
Volume	34
Pages	6003-13
Authors	Jornvall H, Persson B, Krook M, Atrian S, Gonzalez-Duarte R, Jeffery J, Ghosh D
Title	Short-chain dehydrogenases/reductases (SDR).
Related PDB
Related UniProtKB
[2]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) IN COMPLEX WITH NADPH.
Medline ID
PubMed ID	8805511
Journal	Structure
Year	1996
Volume	4
Pages	33-45
Authors	Tanaka N, Nonaka T, Nakanishi M, Deyashiki Y, Hara A, Mitsui Y
Title	Crystal 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 PDB	1cyd
Related UniProtKB	P08074
[3]
Resource
Comments	X-RAY CRYSTALLOGRAPHY
Medline ID
PubMed ID	9271498
Journal	Biochemistry
Year	1997
Volume	36
Pages	10675-84
Authors	Liu Y, Thoden JB, Kim J, Berger E, Gulick AM, Ruzicka FJ, Holden HM, Frey PA
Title	Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli.
Related PDB	1kvu
Related UniProtKB
[4]
Resource
Comments	X-RAY CRYSTALLOGRAPHY
Medline ID
PubMed ID	9817848
Journal	Structure
Year	1998
Volume	6
Pages	1453-65
Authors	Rizzi M, Tonetti M, Vigevani P, Sturla L, Bisso A, Flora AD, Bordo D, Bolognesi M
Title	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.
Related PDB	1bws
Related UniProtKB
[5]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.2 ANGSTROMS)
Medline ID
PubMed ID	9862812
Journal	Structure
Year	1998
Volume	6
Pages	1601-12
Authors	Somers WS, Stahl ML, Sullivan FX
Title	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.
Related PDB	1bsv 1fxs 1gfs
Related UniProtKB	P32055
[6]
Resource
Comments
Medline ID
PubMed ID	10480878
Journal	J Biol Chem
Year	1999
Volume	274
Pages	26743-50
Authors	Menon S, Stahl M, Kumar R, Xu GY, Sullivan F
Title	Stereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli.
Related PDB
Related UniProtKB
[7]
Resource
Comments	X-RAY CRYSTALLOGRAPHY
Medline ID
PubMed ID	11021971
Journal	J Mol Biol
Year	2000
Volume	303
Pages	77-91
Authors	Rosano C, Bisso A, Izzo G, Tonetti M, Sturla L, De Flora A, Bolognesi M
Title	Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.
Related PDB	1e6u 1e7q 1e7r 1e7s
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID	10896473
Journal	Structure
Year	2000
Volume	8
Pages	453-62
Authors	Deacon AM, Ni YS, Coleman WG Jr, Ealick SE
Title	The crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist.
Related PDB	1eq2
Related UniProtKB	P67910
[9]
Resource
Comments
Medline ID
PubMed ID	11706991
Journal	Cell Mol Life Sci
Year	2001
Volume	58
Pages	1650-65
Authors	Allard ST, Giraud MF, Naismith JH
Title	Epimerases: structure, function and mechanism.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID	15023057
Journal	Biochemistry
Year	2004
Volume	43
Pages	3057-67
Authors	Vogan EM, Bellamacina C, He X, Liu HW, Ringe D, Petsko GA
Title	Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID	15823050
Journal	Biochemistry
Year	2005
Volume	44
Pages	5907-15
Authors	Morrison JP, Read JA, Coleman WG Jr, Tanner ME
Title	Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism.
Related PDB
Related UniProtKB
[12]
Resource
Comments	X-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.
Medline ID
PubMed ID	16366586
Journal	J Am Chem Soc
Year	2005
Volume	127
Pages	18309-20
Authors	Major LL, Wolucka BA, Naismith JH
Title	Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.
Related PDB	2c5A 2c5e 2c54 2c59
Related UniProtKB	Q93VR3
[13]
Resource
Comments
Medline ID
PubMed ID	19058170
Journal	Angew Chem Int Ed Engl
Year	2008
Volume	47
Pages	9814-59
Authors	Thibodeaux CJ, Melancon CE 3rd, Liu HW
Title	Natural-product sugar biosynthesis and enzymatic glycodiversification.
Related PDB
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID	19053199
Journal	J Am Chem Soc
Year	2008
Volume	130
Pages	17593-602
Authors	Lau ST, Tanner ME
Title	Mechanism and active site residues of GDP-fucose synthase.
Related PDB
Related UniProtKB

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).

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).

Created	Updated
2010-08-06	2011-08-10