EzCatDB: T00201

DB codeT00201
RLCP classification1.13.30110.56
5.21.14000.74
8.121.42000.7
8.113.46500.7
CATH domainDomain 13.60.20.10Catalytic domain
Domain 23.40.50.10490Catalytic domain
Domain 33.40.50.10490
E.C.2.6.1.16
CSA1gdo,1jxa,1moq
MACiEM0082

CATH domainRelated DB codes (homologues)
3.40.50.10490T00232
3.60.20.10M00123,M00174,D00300

Enzyme Name
Swiss-protKEGG

P17169
Protein nameGlucosamine--fructose-6-phosphate aminotransferase {isomerizing}glutamine---fructose-6-phosphate transaminase (isomerizing)
hexosephosphate aminotransferase
glucosamine-6-phosphate isomerase (glutamine-forming)
glutamine-fructose-6-phosphate transaminase (isomerizing)
D-fructose-6-phosphate amidotransferase
glucosaminephosphate isomerase
glucosamine 6-phosphate synthase
GlcN6P synthase
SynonymsEC 2.6.1.16
Hexosephosphate aminotransferase
D-fructose-6-phosphate amidotransferase
GFAT
L-glutamine-D-fructose-6-phosphate amidotransferase
Glucosamine-6-phosphate synthase

KEGG pathways
MAP codePathways
MAP00251Glutamate metabolism
MAP00530Aminosugars metabolism

Swiss-prot:Accession NumberP17169
Entry nameGLMS_ECOLI
ActivityL-glutamine + D-fructose 6-phosphate = L- glutamate + D-glucosamine 6-phosphate.
SubunitHomodimer.
Subcellular locationCytoplasm.
Cofactor


SubstratesProductsintermediates
KEGG-idC00064C00085C00025C00352
CompoundL-GlutamineD-Fructose 6-phosphateL-GlutamateD-Glucosamine 6-phosphate
Typeamino acids,amide groupcarbohydrate,phosphate group/phosphate ionamino acids,carboxyl groupamine group,carbohydrate,phosphate group/phosphate ion
1gdoAUnboundUnboundBound:GLUUnboundUnbound
1gdoBUnboundUnboundBound:GLUUnboundUnbound
1gdoCUnboundUnboundBound:GLUUnboundUnbound
1gdoDUnboundUnboundBound:GLUUnboundUnbound
1gmsAAnalogue:HGAUnboundUnboundUnboundUnbound
1gmsCAnalogue:HGAUnboundUnboundUnboundUnbound
1gmsEAnalogue:HGAUnboundUnboundUnboundUnbound
1gmsGAnalogue:HGAUnboundUnboundUnboundUnbound
1jxaA01UnboundUnboundUnboundUnboundUnbound
1jxaB01UnboundUnboundUnboundUnboundUnbound
1jxaC01UnboundUnboundUnboundUnboundUnbound
1moqA01UnboundUnboundUnboundBound:GLPUnbound
1morA01UnboundUnboundUnboundAnalogue:G6PUnbound
1mosA01UnboundUnboundUnboundAnalogue:AGPUnbound
1jxaA02UnboundUnboundUnboundUnboundIntermediate-analogue:G6Q
1jxaB02UnboundUnboundUnboundUnboundIntermediate-analogue:G6Q
1jxaC02UnboundUnboundUnboundUnboundIntermediate-analogue:G6Q
1moqA02UnboundUnboundUnboundUnboundUnbound
1morA02UnboundUnboundUnboundUnboundUnbound
1mosA02UnboundUnboundUnboundUnboundUnbound
1jxaA03UnboundUnboundUnboundUnboundUnbound
1jxaB03UnboundUnboundUnboundUnboundUnbound
1jxaC03UnboundUnboundUnboundUnboundUnbound

Active-site residues
resource
Swiss-prot;P17169 & literature [6], [11], [13], [14] & [15]
pdbCatalytic residuesMain-chain involved in catalysis
1gdoACYS 1;ASN 98
GLY 99
1gdoBCYS 1;ASN 98
GLY 99
1gdoCCYS 1;ASN 98
GLY 99
1gdoDCYS 1;ASN 98
GLY 99
1gmsACYS 1;ASN 98
GLY 99
1gmsCCYS 1;ASN 98
GLY 99
1gmsECYS 1;ASN 98
GLY 99
1gmsGCYS 1;ASN 98
GLY 99
1jxaA01CYS 1;ASN 98
GLY 99
1jxaB01CYS 1;ASN 98
GLY 99
1jxaC01CYS 1;ASN 98
GLY 99
1moqA01LYS 603

1morA01LYS 603

1mosA01LYS 603

1jxaA02LYS 603

1jxaB02LYS 603

1jxaC02LYS 603

1moqA02LYS 485;GLU 488;HIS 504

1morA02LYS 485;GLU 488;HIS 504

1mosA02LYS 485;GLU 488;HIS 504

1jxaA03LYS 485;GLU 488;HIS 504

1jxaB03LYS 485;GLU 488;HIS 504

1jxaC03LYS 485;GLU 488;HIS 504


References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[4]Scheme 2A
[6]Fig.6, p.8076
[10]p.1050-1053
[11]Fig.6, Fig.7, p.599-601
[12]Scheme 24
[13]Fig.7
[14]Scheme 2, p.62-64, Scheme 5, p.64-65, Fig.4, p.65-66
[15]Fig.5, Fig.6, p.179-181

references
[1]
CommentsCHARACTERIZATION
PubMed ID3134953
JournalBiochimie
Year1988
Volume70
Pages287-90
AuthorsDutka-Malen S, Mazodier P, Badet B
TitleMolecular cloning and overexpression of the glucosamine synthetase gene from Escherichia coli.
Related Swiss-protP17169
[2]
PubMed ID1396403
JournalEur Biophys J
Year1992
Volume21
Pages137-45
AuthorsTempczyk A, Tarnowska M, Liwo A, Borowski E
TitleA theoretical study of glucosamine synthase. II. Combined quantum and molecular mechanics simulation of sulfhydryl attack on the carboxyamide group.
[3]
PubMed ID7932726
JournalJ Mol Biol
Year1994
Volume242
Pages703-5
AuthorsObmolova G, Badet-Denisot MA, Badet B, Teplyakov A
TitleCrystallization and preliminary X-ray analysis of the two domains of glucosamine-6-phosphate synthase from Escherichia coli.
[4]
PubMed ID7547881
JournalBiochemistry
Year1995
Volume34
Pages11515-20
AuthorsBearne SL, Wolfenden R
TitleGlutamate gamma-semialdehyde as a natural transition state analogue inhibitor of Escherichia coli glucosamine-6-phosphate synthase.
[5]
PubMed ID8554332
JournalArch Biochem Biophys
Year1995
Volume324
Pages391-400
AuthorsCochet O, Badet-Denisot MA, Teillaud JL, Badet B
TitleEpitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase.
[6]
CommentsX-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) OF 1-240
PubMed ID8805567
JournalStructure
Year1996
Volume4
Pages801-10
AuthorsIsupov MN, Obmolova G, Butterworth S, Badet-Denisot MA, Badet B, Polikarpov I, Littlechild JA, Teplyakov A
TitleSubstrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase.
Related PDB1gms,1gdo
Related Swiss-protP17169
[7]
PubMed ID8621700
JournalJ Biol Chem
Year1996
Volume271
Pages3052-7
AuthorsBearne SL
TitleActive site-directed inactivation of Escherichia coli glucosamine-6-phosphate synthase. Determination of the fructose 6-phosphate binding constant using a carbohydrate-based inactivator.
[8]
PubMed ID8990277
JournalArch Biochem Biophys
Year1997
Volume337
Pages129-36
AuthorsBadet-Denisot MA, Fernandez-Herrero LA, Berenguer J, Ooi T, Badet B
TitleCharacterization of L-glutamine:D-fructose-6-phosphate amidotransferase from an extreme thermophile Thermus thermophilus HB8.
[9]
PubMed ID9151973
JournalEur J Biochem
Year1997
Volume245
Pages418-22
AuthorsLeriche C, Badet-Denisot MA, Badet B
TitleAffinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog--evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site.
[10]
CommentsX-RAY CRYSTALLOGRAPHY (1.57 ANGSTROMS) OF 243-608
PubMed ID9739095
JournalStructure
Year1998
Volume6
Pages1047-55
AuthorsTeplyakov A, Obmolova G, Badet-Denisot MA, Badet B, Polikarpov I
TitleInvolvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 A crystal structure of the isomerase domain.
Related PDB1moq,1mor
Related Swiss-protP17169
[11]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS) OF 241-608
PubMed ID10091662
JournalProtein Sci
Year1999
Volume8
Pages596-602
AuthorsTeplyakov A, Obmolova G, Badet-Denisot MA, Badet B
TitleThe mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase.
Related PDB1mos
Related Swiss-protP17169
[12]
PubMed ID10617596
JournalJ Biol Chem
Year2000
Volume275
Pages135-40
AuthorsBearne SL, Blouin C
TitleInhibition of Escherichia coli glucosamine-6-phosphate synthase by reactive intermediate analogues. The role of the 2-amino function in catalysis.
[13]
PubMed ID11700065
JournalJ Mol Biol
Year2001
Volume313
Pages1093-102
AuthorsTeplyakov A, Obmolova G, Badet B, Badet-Denisot MA
TitleChanneling of ammonia in glucosamine-6-phosphate synthase.
Related PDB1jxa
[14]
PubMed ID11902440
JournalNat Prod Rep
Year2002
Volume19
Pages60-9
AuthorsTeplyakov A, Leriche C, Obmolova G, Badet B, Badet-Denisot MA
TitleFrom Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase.
[15]
PubMed ID12044898
JournalBiochim Biophys Acta
Year2002
Volume1597
Pages173-92
AuthorsMilewski S
TitleGlucosamine-6-phosphate synthase--the multi-facets enzyme.

comments
This enzyme is composed of three domains, N-terminal glutaminase domain, and C-terminal isomerase domains. These domains catalyzes the following reactions:
(A) Hydrolysis of glutamine amide (by the N-terminal glutaminase domain) (see [6] & [14]):
(A#) Binding of the other substrate, Fructose-6-phosphate, to the isomerase domains raise the activity of this catalytic reaction. (see [13])
(A1) Nucleophile, Cys1 can be either in an active conformation with the thiol group close to the substrate amide, or in an inactive one with the group pointing away from the substrate. During the catalysis, its conformation must be active.
(A2) The N-terminal alpha-amino group acts as a general base, which activates the thiol group of the N-terminal Cys1, through a water molecule.
(A3) The activated thiol group attacks the amide carbon of a substrate, glutamine, to form a tetrahedral intermediate. The intermediate is stabilized by an oxyanion hole, made up by sidechain amide of Asn98 and mainchain amide groups of Gly99.
(A4) The intermediate collapses to form a gamma-glutamylthioester and to release ammonia which might be protonated by the water bound to the alpha-amino group of Cys1.
(A5) Another water (distinct from the above bound water) is activated through the bound water by the alpha-amino group.
(A6) The activated water makes a nucleophilic attack on the thiolester carbon, to form a tetrahedral intermediate. This intermediate is also stabilized by the oxyanion hole.
(A7) The intermediate collapses to release the product, glutamate, from Cys1, and the thiol group is protonated through the bound water.
(B) Ring opening of D-Fructose 6-phosphate (Intramolecular elimination) by the isomerase domain (see [11] & [14]):
(B1) His504 (from the adjacent subunit) acts as a general base to deprotonate the O2 hydrogen of the cyclic substrate, D-Fructose 6-phosphate, leading to the formation of carbonyl group and to the cleavage of C2-O5 bond.
(B2) His504 then acts as a general acid to protonate the O5 oxygen.
(C) Schiff-base formation (dehydration) by Lys603:
Detailed mechanism has not been elucidated.
(D) Exchange of Schiff-base nitrogen atoms (by ammonia):
(E) Isomerization (change in position of the double-bond) (see [11], [14] & [15]):
(E1) Glu488 acts as a general base to abstract the pro-R hydrogen from the C1 carbon of the Schiff-base intermediate, leading to the formation of a negatively charged enolamine intermediate. The negatively charged intermediate might be stabilized by Lys603, which has a positive charge.
(E2) Lys603 might protonate the negatively charged nitrogen atom.
(F) Isomerization (change in position of the double-bond):
(F1) Lys485 acts as a general base to abstract proton from the O1 of the enolamine intermediate, whilst Glu488 acts as a general acid to protonate the C2 atom at the same re-face, leading to the formation of C1 carbonyl group.
(#) Chanelling of ammonia from the N-terminal domain to the isomerase domains is also essential to couple the (A) and (D) reactions.

createdupdated
2002-11-292009-02-26


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 2010)
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)

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