DB code: T00201

RLCP classification 1.13.30110.56 : Hydrolysis
5.21.14000.74 : Elimination
8.121.42000.7 : Isomerization
8.113.46500.7 : Isomerization
CATH domain 3.60.20.10 : Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 Catalytic domain
3.40.50.10490 : Rossmann fold Catalytic domain
3.40.50.10490 : Rossmann fold
E.C. 2.6.1.16
CSA 1gdo 1jxa 1moq
M-CSA 1gdo 1jxa 1moq
MACiE M0082

CATH domain Related DB codes (homologues)
3.40.50.10490 : Rossmann fold T00232
3.60.20.10 : Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 M00123 M00174 D00300

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P17169 Glucosamine--fructose-6-phosphate aminotransferase {isomerizing}
EC 2.6.1.16
Hexosephosphate aminotransferase
D-fructose-6-phosphate amidotransferase
GFAT
L-glutamine-D-fructose-6-phosphate amidotransferase
Glucosamine-6-phosphate synthase
NP_418185.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_491700.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
PF00310 (GATase_2)
PF01380 (SIS)
[Graphical View]

KEGG enzyme name
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

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P17169 GLMS_ECOLI L-glutamine + D-fructose 6-phosphate = L- glutamate + D-glucosamine 6-phosphate. Homodimer. Cytoplasm.

KEGG Pathways
Map code Pathways E.C.
MAP00251 Glutamate metabolism
MAP00530 Aminosugars metabolism

Compound table
Substrates Products Intermediates
KEGG-id C00064 C00085 C00025 C00352
E.C.
Compound L-Glutamine D-Fructose 6-phosphate L-Glutamate D-Glucosamine 6-phosphate
Type amino acids,amide group carbohydrate,phosphate group/phosphate ion amino acids,carboxyl group amine group,carbohydrate,phosphate group/phosphate ion
ChEBI 18050
 58359
 18050
 58359
 		61553
 61553
 		16015
 16015
 		47987
 47987
PubChem 5961
 6992086
 5961
 6992086
 		439160
 439160
 		33032
 44272391
 88747398
 33032
 44272391
 88747398
 		440997
 440997
1gdoA Unbound Unbound Bound:GLU Unbound Unbound
1gdoB Unbound Unbound Bound:GLU Unbound Unbound
1gdoC Unbound Unbound Bound:GLU Unbound Unbound
1gdoD Unbound Unbound Bound:GLU Unbound Unbound
1gmsA Analogue:HGA Unbound Unbound Unbound Unbound
1gmsC Analogue:HGA Unbound Unbound Unbound Unbound
1gmsE Analogue:HGA Unbound Unbound Unbound Unbound
1gmsG Analogue:HGA Unbound Unbound Unbound Unbound
1jxaA01 Unbound Unbound Unbound Unbound Unbound
1jxaB01 Unbound Unbound Unbound Unbound Unbound
1jxaC01 Unbound Unbound Unbound Unbound Unbound
1moqA01 Unbound Unbound Unbound Bound:GLP Unbound
1morA01 Unbound Unbound Unbound Analogue:G6P Unbound
1mosA01 Unbound Unbound Unbound Analogue:AGP Unbound
1jxaA02 Unbound Unbound Unbound Unbound Intermediate-analogue:G6Q
1jxaB02 Unbound Unbound Unbound Unbound Intermediate-analogue:G6Q
1jxaC02 Unbound Unbound Unbound Unbound Intermediate-analogue:G6Q
1moqA02 Unbound Unbound Unbound Unbound Unbound
1morA02 Unbound Unbound Unbound Unbound Unbound
1mosA02 Unbound Unbound Unbound Unbound Unbound
1jxaA03 Unbound Unbound Unbound Unbound Unbound
1jxaB03 Unbound Unbound Unbound Unbound Unbound
1jxaC03 Unbound Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;P17169 & literature [6], [11], [13], [14] & [15]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1gdoA CYS 1;ASN 98 GLY 99
1gdoB CYS 1;ASN 98 GLY 99
1gdoC CYS 1;ASN 98 GLY 99
1gdoD CYS 1;ASN 98 GLY 99
1gmsA CYS 1;ASN 98 GLY 99
1gmsC CYS 1;ASN 98 GLY 99
1gmsE CYS 1;ASN 98 GLY 99
1gmsG CYS 1;ASN 98 GLY 99
1jxaA01 CYS 1;ASN 98 GLY 99
1jxaB01 CYS 1;ASN 98 GLY 99
1jxaC01 CYS 1;ASN 98 GLY 99
1moqA01 LYS 603
1morA01 LYS 603
1mosA01 LYS 603
1jxaA02 LYS 603
1jxaB02 LYS 603
1jxaC02 LYS 603
1moqA02 LYS 485;GLU 488;HIS 504
1morA02 LYS 485;GLU 488;HIS 504
1mosA02 LYS 485;GLU 488;HIS 504
1jxaA03 LYS 485;GLU 488;HIS 504
1jxaB03 LYS 485;GLU 488;HIS 504
1jxaC03 LYS 485;GLU 488;HIS 504

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[4]
Scheme 2A
[6]
Fig.6, p.807 6
[10]
p.1050-1053
[11]
Fig.6, Fig.7, p.599-601
[12]
Scheme 2 4
[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]
Resource
Comments CHARACTERIZATION
Medline ID
PubMed ID 3134953
Journal Biochimie
Year 1988
Volume 70
Pages 287-90
Authors Dutka-Malen S, Mazodier P, Badet B
Title Molecular cloning and overexpression of the glucosamine synthetase gene from Escherichia coli.
Related PDB
Related UniProtKB P17169
[2]
Resource
Comments
Medline ID
PubMed ID 1396403
Journal Eur Biophys J
Year 1992
Volume 21
Pages 137-45
Authors Tempczyk A, Tarnowska M, Liwo A, Borowski E
Title A theoretical study of glucosamine synthase. II. Combined quantum and molecular mechanics simulation of sulfhydryl attack on the carboxyamide group.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 7932726
Journal J Mol Biol
Year 1994
Volume 242
Pages 703-5
Authors Obmolova G, Badet-Denisot MA, Badet B, Teplyakov A
Title Crystallization and preliminary X-ray analysis of the two domains of glucosamine-6-phosphate synthase from Escherichia coli.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 7547881
Journal Biochemistry
Year 1995
Volume 34
Pages 11515-20
Authors Bearne SL, Wolfenden R
Title Glutamate gamma-semialdehyde as a natural transition state analogue inhibitor of Escherichia coli glucosamine-6-phosphate synthase.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 8554332
Journal Arch Biochem Biophys
Year 1995
Volume 324
Pages 391-400
Authors Cochet O, Badet-Denisot MA, Teillaud JL, Badet B
Title Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) OF 1-240
Medline ID
PubMed ID 8805567
Journal Structure
Year 1996
Volume 4
Pages 801-10
Authors Isupov MN, Obmolova G, Butterworth S, Badet-Denisot MA, Badet B, Polikarpov I, Littlechild JA, Teplyakov A
Title Substrate 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 PDB 1gms 1gdo
Related UniProtKB P17169
[7]
Resource
Comments
Medline ID
PubMed ID 8621700
Journal J Biol Chem
Year 1996
Volume 271
Pages 3052-7
Authors Bearne SL
Title Active site-directed inactivation of Escherichia coli glucosamine-6-phosphate synthase. Determination of the fructose 6-phosphate binding constant using a carbohydrate-based inactivator.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 8990277
Journal Arch Biochem Biophys
Year 1997
Volume 337
Pages 129-36
Authors Badet-Denisot MA, Fernandez-Herrero LA, Berenguer J, Ooi T, Badet B
Title Characterization of L-glutamine:D-fructose-6-phosphate amidotransferase from an extreme thermophile Thermus thermophilus HB8.
Related PDB
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID 9151973
Journal Eur J Biochem
Year 1997
Volume 245
Pages 418-22
Authors Leriche C, Badet-Denisot MA, Badet B
Title Affinity 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.
Related PDB
Related UniProtKB
[10]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.57 ANGSTROMS) OF 243-608
Medline ID
PubMed ID 9739095
Journal Structure
Year 1998
Volume 6
Pages 1047-55
Authors Teplyakov A, Obmolova G, Badet-Denisot MA, Badet B, Polikarpov I
Title Involvement 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 PDB 1moq 1mor
Related UniProtKB P17169
[11]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS) OF 241-608
Medline ID
PubMed ID 10091662
Journal Protein Sci
Year 1999
Volume 8
Pages 596-602
Authors Teplyakov A, Obmolova G, Badet-Denisot MA, Badet B
Title The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase.
Related PDB 1mos
Related UniProtKB P17169
[12]
Resource
Comments
Medline ID
PubMed ID 10617596
Journal J Biol Chem
Year 2000
Volume 275
Pages 135-40
Authors Bearne SL, Blouin C
Title Inhibition of Escherichia coli glucosamine-6-phosphate synthase by reactive intermediate analogues. The role of the 2-amino function in catalysis.
Related PDB
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID 11700065
Journal J Mol Biol
Year 2001
Volume 313
Pages 1093-102
Authors Teplyakov A, Obmolova G, Badet B, Badet-Denisot MA
Title Channeling of ammonia in glucosamine-6-phosphate synthase.
Related PDB 1jxa
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID 11902440
Journal Nat Prod Rep
Year 2002
Volume 19
Pages 60-9
Authors Teplyakov A, Leriche C, Obmolova G, Badet B, Badet-Denisot MA
Title From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 12044898
Journal Biochim Biophys Acta
Year 2002
Volume 1597
Pages 173-92
Authors Milewski S
Title Glucosamine-6-phosphate synthase--the multi-facets enzyme.
Related PDB
Related UniProtKB

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.

Created Updated
2002-11-29 2009-02-26