EzCatDB: D00262
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DB codeD00262
RLCP classification9.1050.440000.8011 : Hydride transfer
5.10.9510.969 : Elimination
9.5010.536210.8011 : Hydride transfer
CATH domainDomain 13.40.50.720 : Rossmann foldCatalytic domain
Domain 23.90.25.10 : UDP-galactose 4-epimerase; domain 1
E.C.4.2.1.46
MACiEM0228

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,D00604,D00605,D00615,D00845,D00857,D00858,M00161,M00171,M00210,T00002,T00010,T00011,T00015,T00227,T00247,T00408,T00414,D00827,D00274,D00275,M00035,T00109
3.90.25.10 : UDP-galactose 4-epimerase; domain 1D00513,D00601,D00604,D00274,D00275

Enzyme Name
UniProtKBKEGG

P27830Q9EU31P26391P95780Q8GIP9Q9ZGH3
Protein namedTDP-glucose 4,6-dehydratase 2dTDP-glucose 4,6-dehydratasedTDP-glucose 4,6-dehydratasedTDP-glucose 4,6-dehydratasedTDP-glucose 4,6-dehydratasedTDP-glucose 4,6-dehydratasedTDP-glucose 4,6-dehydratase
thymidine diphosphoglucose oxidoreductase
TDP-glucose oxidoreductase
dTDP-glucose 4,6-hydro-lyase
SynonymsEC 4.2.1.46
EC 4.2.1.46
EC 4.2.1.46
EC 4.2.1.46
EC 4.2.1.46
EC 4.2.1.46
RefSeqYP_026255.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_491651.1 (Protein)
NC_007779.1 (DNA/RNA sequence)

NP_461042.1 (Protein)
NC_003197.1 (DNA/RNA sequence)
NP_721810.1 (Protein)
NC_004350.2 (DNA/RNA sequence)
YP_004401333.1 (Protein)
NC_015433.1 (DNA/RNA sequence)

PfamPF01370 (Epimerase)
[Graphical view]
PF01370 (Epimerase)
[Graphical view]
PF01370 (Epimerase)
[Graphical view]
PF01370 (Epimerase)
[Graphical view]
PF01370 (Epimerase)
[Graphical view]
PF01370 (Epimerase)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00520Nucleotide sugars metabolism
MAP00521Streptomycin biosynthesis
MAP00523Polyketide sugar unit biosynthesis
MAP01055Biosynthesis of vancomycin group antibiotics

UniProtKB:Accession NumberP27830Q9EU31P26391P95780Q8GIP9Q9ZGH3
Entry nameRFFG_ECOLIQ9EU31_SALCHRMLB_SALTYRMLB_STRMUQ8GIP9_STRSUQ9ZGH3_9ACTO
ActivitydTDP-glucose = dTDP-4-dehydro-6-deoxy-D-glucose + H(2)O.dTDP-glucose = dTDP-4-dehydro-6-deoxy-D-glucose + H(2)O.dTDP-glucose = dTDP-4-dehydro-6-deoxy-D-glucose + H(2)O.dTDP-glucose = dTDP-4-dehydro-6-deoxy-D-glucose + H(2)O.dTDP-glucose = dTDP-4-dehydro-6-deoxy-D-glucose + H(2)O.dTDP-glucose = dTDP-4-dehydro-6-deoxy-D-glucose + H(2)O.
SubunitHomodimer.
Homodimer.Homodimer.

Subcellular location





CofactorNAD.NAD (By similarity).Binds 1 NAD ion per monomer.Binds 1 NAD ion per monomer.NAD (By similarity).NAD (By similarity).

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC00003C00842C11907C00001I00091I00092
CompoundNAD+dTDP-glucosedTDP-4-dehydro-6-deoxy-D-glucoseH2OdTDP-4-dehydro-glucosedTDP-6-deoxy-4-dehydro-5,6-ene-glucose
Typeamide group,amine group,nucleotideamide group,carbohydrate,nucleotideamide group,carbohydrate,nucleotideH2O

ChEBI15846
15700
16128
15377


PubChem5893
443210
439292
22247451
962


              
1bxkA01Bound:NADUnboundUnbound UnboundUnbound
1bxkB01Bound:NADUnboundUnbound UnboundUnbound
1g1aA01Bound:NADUnboundUnbound UnboundUnbound
1g1aB01Bound:NADUnboundUnbound UnboundUnbound
1g1aC01Bound:NADUnboundUnbound UnboundUnbound
1g1aD01Bound:NADUnboundUnbound UnboundUnbound
1keuA01Bound:NADUnboundUnbound UnboundUnbound
1keuB01Bound:NADUnboundUnbound UnboundUnbound
1kewA01Bound:NADUnboundUnbound UnboundUnbound
1kewB01Bound:NADUnboundUnbound UnboundUnbound
1kepA01Bound:NADAnalogue:TDXUnbound UnboundUnbound
1kepB01Bound:NADUnboundUnbound UnboundUnbound
1kerA01Bound:NADUnboundUnbound UnboundUnbound
1kerB01Bound:NADUnboundUnbound UnboundUnbound
1ketA01Bound:NADUnboundUnbound UnboundUnbound
1ketB01Bound:NADUnboundUnbound UnboundUnbound
1oc2A01Bound:NADUnboundUnbound UnboundUnbound
1oc2B01Bound:NADUnboundUnbound UnboundUnbound
1r66A01Bound:NADUnboundUnbound UnboundUnbound
1r6dA01Bound:NADUnboundUnbound UnboundUnbound
1bxkA02UnboundUnboundUnbound UnboundUnbound
1bxkB02UnboundUnboundUnbound UnboundUnbound
1g1aA02UnboundUnboundUnbound UnboundUnbound
1g1aB02UnboundUnboundUnbound UnboundUnbound
1g1aC02UnboundUnboundUnbound UnboundUnbound
1g1aD02UnboundUnboundUnbound UnboundUnbound
1keuA02UnboundBound:DAUUnbound UnboundUnbound
1keuB02UnboundBound:DAUUnbound UnboundUnbound
1kewA02UnboundAnalogue:TYDUnbound UnboundUnbound
1kewB02UnboundAnalogue:TYDUnbound UnboundUnbound
1kepA02UnboundUnboundUnbound UnboundUnbound
1kepB02UnboundAnalogue:TDXUnbound UnboundUnbound
1kerA02UnboundBound:DAUUnbound UnboundUnbound
1kerB02UnboundBound:DAUUnbound UnboundUnbound
1ketA02UnboundAnalogue:TYDUnbound UnboundUnbound
1ketB02UnboundAnalogue:TYDUnbound UnboundUnbound
1oc2A02UnboundAnalogue:TDXUnbound UnboundUnbound
1oc2B02UnboundAnalogue:TDXUnbound UnboundUnbound
1r66A02UnboundAnalogue:TYDUnbound UnboundUnbound
1r6dA02UnboundBound:DAUUnbound UnboundUnbound

Active-site residues
resource
literature [8], [9], [12], [13] & [14]
pdbCatalytic residuescomment
          
1bxkA01THR 134;ASP 135;GLU 136;TYR 160;LYS 164;ARG 224
 
1bxkB01THR 534;ASP 535;GLU 536;TYR 560;LYS 564;ARG 624
 
1g1aA01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1g1aB01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1g1aC01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1g1aD01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1keuA01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1keuB01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1kewA01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1kewB01THR 133;ASP 134;GLU 135;TYR 167;LYS 171;ARG 231
 
1kepA01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1kepB01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1kerA01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1kerB01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1ketA01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1ketB01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1oc2A01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1oc2B01THR 125;ASP 126;GLU 127;TYR 161;LYS 165;ARG 225
 
1r66A01THR 127;ASP 128;GLU 129;TYR 151;LYS 155;ARG 215
 
1r6dA01THR 127;       ;       ;TYR 151;LYS 155;ARG 215
mutant D128N, E129Q
1bxkA02 
 
1bxkB02 
 
1g1aA02 
 
1g1aB02 
 
1g1aC02 
 
1g1aD02 
 
1keuA02 
 
1keuB02 
 
1kewA02 
 
1kewB02 
 
1kepA02 
 
1kepB02 
 
1kerA02 
 
1kerB02 
 
1ketA02 
 
1ketB02 
 
1oc2A02 
 
1oc2B02 
 
1r66A02 
 
1r6dA02 
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[7]Scheme 1, Fig.1, Fig.52
[8]Scheme 1, Scheme 2, Scheme 32
[9]Scheme 1, Scheme 2, Scheme 32
[10]Scheme 1, Scheme 3, Scheme 4, Scheme 53
[12]Fig.15
[13]Scheme 1, Scheme 2, Fig.4, Scheme 3
[14]Fig.4, p.883
[15]Scheme 1
[16]Scheme 2, p.2217-2220
[17]Fig.3, p.648-651

references
[1]
PubMed ID7517391
JournalJ Bacteriol
Year1994
Volume176
Pages4144-56
AuthorsStevenson G, Neal B, Liu D, Hobbs M, Packer NH, Batley M, Redmond JW, Lindquist L, Reeves P
TitleStructure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.
[2]
CommentsFUNCTION
Medline ID96032389
PubMed ID7559340
JournalJ Bacteriol
Year1995
Volume177
Pages5539-46
AuthorsMarolda CL, Valvano MA
TitleGenetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene.
Related UniProtKBP27830
[3]
PubMed ID9011374
JournalCarbohydr Res
Year1996
Volume285
Pages141-50
AuthorsNaundorf A, Klaffke W
TitleSubstrate specificity of native dTDP-D-glucose-4,6-dehydratase: chemo-enzymatic syntheses of artificial and naturally occurring deoxy sugars.
[4]
PubMed ID10462438
JournalArch Biochem Biophys
Year1999
Volume369
Pages30-41
AuthorsEssigmann B, Hespenheide BM, Kuhn LA, Benning C
TitlePrediction of the active-site structure and NAD(+) binding in SQD1, a protein essential for sulfolipid biosynthesis in Arabidopsis.
[5]
PubMed ID10358040
JournalJ Biol Chem
Year1999
Volume274
Pages16933-9
AuthorsYoshida Y, Nakano Y, Nezu T, Yamashita Y, Koga T
TitleA novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.
[6]
PubMed ID10666612
JournalActa Crystallogr D Biol Crystallogr
Year2000
Volume56
Pages222-5
AuthorsAllard ST, Giraud MF, Whitfield C, Messner P, Naismith JH
TitleThe purification, crystallization and structural elucidation of dTDP-D-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-L-rhamnose synthesis pathway from Salmonella enterica serovar typhimurium.
[7]
PubMed ID11076501
JournalBiochemistry
Year2000
Volume39
Pages13633-40
AuthorsGross JW, Hegeman AD, Vestling MM, Frey PA
TitleCharacterization of enzymatic processes by rapid mix-quench mass spectrometry: the case of dTDP-glucose 4,6-dehydratase.
[8]
PubMed ID11478886
JournalBiochemistry
Year2001
Volume40
Pages9187-95
AuthorsGerratana B, Cleland WW, Frey PA
TitleMechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase.
[9]
PubMed ID11601973
JournalBiochemistry
Year2001
Volume40
Pages12497-504
AuthorsGross JW, Hegeman AD, Gerratana B, Frey PA
TitleDehydration is catalyzed by glutamate-136 and aspartic acid-135 active site residues in Escherichia coli dTDP-glucose 4,6-dehydratase.
[10]
PubMed ID11380254
JournalBiochemistry
Year2001
Volume40
Pages6598-610
AuthorsHegeman AD, Gross JW, Frey PA
TitleProbing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site.
[11]
PubMed ID11553351
JournalCarbohydr Res
Year2001
Volume335
Pages23-32
AuthorsAmann S, Drager G, Rupprath C, Kirschning A, Elling L
Title(Chemo)enzymatic synthesis of dTDP-activated 2,6-dideoxysugars as building blocks of polyketide antibiotics.
[12]
CommentsX-ray crystallography
PubMed ID11243820
JournalJ Mol Biol
Year2001
Volume307
Pages283-95
AuthorsAllard ST, Giraud MF, Whitfield C, Graninger M, Messner P, Naismith JH
TitleThe crystal structure of dTDP-D-Glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar Typhimurium, the second enzyme in the dTDP-l-rhamnose pathway.
Related PDB1g1a
[13]
PubMed ID11851427
JournalBiochemistry
Year2002
Volume41
Pages2797-804
AuthorsHegeman AD, Gross JW, Frey PA
TitleConcerted and stepwise dehydration mechanisms observed in wild-type and mutated Escherichia coli dTDP-glucose 4,6-dehydratase.
[14]
PubMed ID11796113
JournalStructure (Camb)
Year2002
Volume10
Pages81-92
AuthorsAllard ST, Beis K, Giraud MF, Hegeman AD, Gross JW, Wilmouth RC, Whitfield C, Graninger M, Messner P, Allen AG, Maskell DJ, Naismith JH
TitleToward a structural understanding of the dehydratase mechanism.
Related PDB1kep,1ker,1keu,1ket,1kew
[15]
PubMed ID14505409
JournalJ Am Chem Soc
Year2003
Volume125
Pages11872-8
AuthorsBeis K, Allard ST, Hegeman AD, Murshudov G, Philp D, Naismith JH
TitleThe structure of NADH in the enzyme dTDP-d-glucose dehydratase (RmlB).
Related PDB1oc2
[16]
PubMed ID14570895
JournalJ Biol Chem
Year2004
Volume279
Pages2211-20
AuthorsAllard ST, Cleland WW, Holden HM
TitleHigh resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae.
Related PDB1r66,1r6d
[17]
PubMed ID15493979
JournalBiochem Soc Trans
Year2004
Volume32
Pages647-54
AuthorsNaismith JH
TitleChemical insights from structural studies of enzymes.

comments
This enzyme belongs to the Short-chain dehydrogenases/reductases (SDR).
Although the catalytic site is slightly different, this enzyme is homologous to the GDP-mannose 4,6-dehydratases (EC 4.2.1.47, D00513 and D00543 in EzCatDB).
This enzyme catalyzes reactions similar to those by its homologous enzymes (D00513 and D00543) (see [14]).
According to the literature [14], [16] and [17], this enzyme catalyzes at least three reactions: oxidation of dTDP-glucose (hydride transfer from dTDP-glucose to nicotinamide), dehydration (elimination of a hydroxyl group from C6), and rereduction of C5-C6 double bond to methyl group (hydride transfer from nicotinamide to the intermediate).
Taken together, this enzyme catalyzes the following reactions:
(A) Hydride transfer from C4 atom of substrate to NAD, forming a 4-keto intermediate (I00091):
(A0) Lys171 (of 1g1a) modulates the activity (or pKa) of Tyr167 via 2'-hydroxyl group of NAD, along with the N1 atom of the nicotinamide group in NAD, whereas Thr133 modulates the pKa of 4-hydroxyl oxygen of the substrate.
(A1) Tyr167 acts as a general base to deprotonate the 4-hydroxyl oxygen of the substrate. Meanwhile, the hydride transfer occurs from the C4-carbon of the substrate to the C4 atom of the nicotinamide, forming the 4-keto intermediate (I00091).
(B) Elimination of hydroxyl group from C6 of the intermediate, forming 4-keto-5,6-glucosen intermediate (I00092):
(B1) Glu135 acts as a general base to deprotonate the C5 atom, possibly forming an enol/enolate transition-state. This transition-state might be stabilized by Tyr167 and Thr133.
(B2) Asp134 acts as a general acid to protonate the O6 hydroxyl group, to release a water molecule, and to form the 4-keto-5,6-ene intermediate from the enol/enolate transition-state.
(C) Hydride transfer from NADH to C6 atom of the intermediate (I00092):
(C0) A slight rotation of the hexose ring of intermediate might be necessary for the reaction. Lys171 modulates the activity (or pKa) of Tyr167 via 2'-hydroxyl group of NAD, along with the N1 atom of the nicotinamide group in NAD.
(C1) Hydride transfer from NADH to C6 atom of the hexose ring in the intermediate. Meanwhile, a general acid must protonate the C5 atom of the hexose. According to the literature [14] and [17], Tyr167 seems to play a role as a general acid. (Thr133 might assist Tyr167 as a general acid, probably acting as a proton shuttle as in the homologous enzyme.)

createdupdated
2004-06-072011-06-16
Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
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)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
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