M-CSA Mechanism and Catalytic Site Atlas

Glutamine-fructose-6-phosphate transaminase (isomerizing)

Glucosamine-fructose-6-phosphate aminotransferase (isomerizing) also known as Glucosamine-6-phosphate synthase (GlmS), catalyses the first reaction in hexamine biosynthesis. It belongs to an F-type group of glutamine-dependent amidotransferase family of enzymes, which utilise the glutamine amide nitrogen in the biosynthesis of phosphoribosylamine, glutamate or asparagine.

The hexosamine biosynthetic pathway starts from D fructose-6-phosphate (Fru6P)2 which is produced from glucose via the glycolysis pathway. The Fru6P is converted into D -glucosamine-6-phosphate (GlcN6P) by the rate-limiting enzyme glucosamine-6-phosphate synthase (GlcN6P synthase). This is the sole biosynthetic route to GlcN6P known to date.

The reaction is practically irreversible and the reaction takes place over two structural domains, an N-terminal glutaminase domain, which hydrolyses glutamine to glutamate and ammonia (residues 1-240), and a C-terminal isomerase domain (residues 241-608), which catalyses the ketose-aldose isomerisation and utilises the nitrogen for synthesis of GlcN-6P.

The isomerase domain is responsible for two activities of GlmS, the conversion of Fru-6P into GlcN-6P in the presence of glutamine (the synthase activity), and the isomerisation of Fru-6P into Glc-6P (the phosphoglucose isomerase - like activity) in the absence of glutamine.

The product of the reaction with fructose 6-phosphate, glucosamine 6-phosphate, undergoes transformation leading towards formation of uridine diphospho-N-acetylglucosamine - which is a precursor to all amino sugar-containing macromolecules. Much interest has been shown in this enzyme that is believed to have important implications in antibacterial/antifungal therapy and diabetes treatment.

Reference Protein and Structure

Sequence: P17169 (2.6.1.16) (Sequence Homologues) (PDB Homologues)
Biological species: Escherichia coli K-12 (Bacteria)
PDB: 1jxa - GLUCOSAMINE 6-PHOSPHATE SYNTHASE WITH GLUCOSE 6-PHOSPHATE (3.1 Å)
Catalytic CATH Domains: 3.60.20.10 3.40.50.10490 (see all for 1jxa)
Cofactors: Water (1)

Click To Show Structure

Enzyme Reaction (EC:2.6.1.16)

D-fructofuranose 6-phosphate(2-)

CHEBI:61527

L-glutamine zwitterion

CHEBI:58359

→

L-glutamate(1-)

CHEBI:29985

2-ammonio-2-deoxy-D-glucopyranose 6-phosphate(1-)

CHEBI:58725

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: D-fructose-6-phosphate amidotransferase, GlcN6P synthase, Glucosamine 6-phosphate synthase, Glucosamine-6-phosphate isomerase (glutamine-forming), Glucosaminephosphate isomerase, Hexosephosphate aminotransferase, Glucosamine-6-phosphate synthase, L-glutamine-D-fructose-6-phosphate amidotransferase,

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

Summary
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
Step 9
Step 10
Step 11
Step 12
Step 13
Step 14
Products
All Steps

Introduction

In the GAT domain deprotonated Cys1 attacks the amide carbonyl of L-glutamine forming a tetrahedral intermediate. Gly99 and Asn98 stabilise negative charge by formation of an oxyanion hole via hydrogen bonding. Subsequent collapse leads to release of ammonia, which is then transferred to the isomerase domain via a channel. In the GAT domain a deprotonated water molecule then attacks the carbonyl group to form a second tetrahedral intermediate with collapse leading to release of Cys1. Note during the reaction in GAT domain the base that deprotonates water is the N-terminus of Cys1.

In the isomerase domain His504 deprotonates the hydroxyl group of C2, which leads to ring opening of Fru6P. Then Lys603 forms an imine linkage with Fru6P via nucleophilic attack. Ammonia formed in the GAT domain then attacks this linkage leading to nucleophilic substituion. Glu488 deprotonates C1 with concommitant double bond rearrangement that deprotonates Lys485. Lys485 then deprotonates C1-OH with concommitant double bond rearrangement and deprotonation of Glu488. Finally His504 deprotonates C5 hydroxyl,the resulting oxyanion initiates a nucleophilic attack on the C1 carbonyl carbon in an addition reaction with concomitant deprotonation of His504B leading to formation of the GlcN6P product.

Catalytic Residues Roles

UniProt	PDB* (1jxa)
Glu482	Glu481A	Stabilises and activates the catalytic lysine (Lys485).	hydrogen bond acceptor, electrostatic stabiliser
Cys2	Cys1A	Acts as the catalytic nucleophile in the deamination reaction. It is activated by its own main chain amide (which is in the form of the N-terminus of the protein).	covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
Trp75 (main-N), Arg27 (main-C)	Trp74A (main-N), Arg26A (main-C)	Stabilise the formation of protonated N-terminus of Cys1 via hydrogen bonding.	hydrogen bond donor, electrostatic stabiliser
Cys2 (N-term)	Cys1A (N-term)	N-terminus acts as a base to deprotonate water, which in turn deprotonates the thiol proton to form a nucleophile. When ammonia leaves it picks up a proton from the N-terminus. The N-terminus then deprotonates another molecule of water to form a hydroxide nucleophile.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu489	Glu488A	Acts as a general acid/base in the deprotonation of C1 to form the isomer.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys604	Lys603A	Acts as a nucleophile to form imine linkage with carbonyl group.	covalently attached, hydrogen bond donor, nucleofuge, proton acceptor, proton donor, nucleophile, electron pair acceptor, electron pair donor
Lys486	Lys485A	Acts as a general acid/base in the isomerisation of Fru6p to Glc6P.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Gly100 (main-N), Asn99	Gly99A (main-N), Asn98A	Forms a hydrogen bond to the negatively charged oxygen to stabilise its formation, via an oxyanion hole.	hydrogen bond donor, electrostatic stabiliser
His505	His504A(AA)	Acts as a general base to initiate decyclisation of Fru6P. Then protonates in the final step to form Glc6P.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton relay, unimolecular elimination by the conjugate base, deamination, enzyme-substrate complex cleavage, intermediate collapse, overall product formed, native state of enzyme regenerated, intermediate terminated, bimolecular elimination, decyclisation, dehydration, schiff base formed, intramolecular elimination, assisted tautomerisation (not keto-enol), assisted keto-enol tautomerisation, intramolecular nucleophilic addition, cyclisation

References

Durand P et al. (2008), Arch Biochem Biophys, 474, 302-317. Highlights of glucosamine-6P synthase catalysis. DOI:10.1016/j.abb.2008.01.026. PMID:18279655.
Chennu MM et al. (2015), Bioinformation, 11, 525-528. Molecular docking based screening of G6PS with 1, 5 Benzothiazepine derivates for a potential inhibitor. DOI:10.6026/97320630011525. PMID:26770025.
Jędrzejczak R et al. (2012), Chembiochem, 13, 85-96. Inactivation of Glucosamine-6-Phosphate Synthase by N3-Oxoacyl Derivatives of L-2,3-Diaminopropanoic Acid. DOI:10.1002/cbic.201100587. PMID:22125025.
Mouilleron S et al. (2011), Arch Biochem Biophys, 505, 1-12. Dynamics of glucosamine-6-phosphate synthase catalysis. DOI:10.1016/j.abb.2010.08.008. PMID:20709015.
Floquet N et al. (2009), J Mol Biol, 385, 653-664. Collective motions in Glucosamine-6-phosphate Synthase: Influence of Ligand Binding and role in Ammonia Channelling and Opening of the Fructose-6-Phosphate Binding Site. DOI:10.1016/j.jmb.2008.10.032. PMID:18976669.
Mouilleron S et al. (2008), J Mol Biol, 377, 1174-1185. Ordering of C-terminal Loop and Glutaminase Domains of Glucosamine-6-Phosphate Synthase Promotes Sugar Ring Opening and Formation of the Ammonia Channel. DOI:10.1016/j.jmb.2008.01.077. PMID:18295797.
Mouilleron S et al. (2007), Protein Sci, 16, 485-493. Domain motions of glucosamine-6P synthase: Comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation. DOI:10.1110/ps.062598107. PMID:17322533.
Mouilleron S et al. (2006), J Biol Chem, 281, 4404-4412. Glutamine Binding Opens the Ammonia Channel and Activates Glucosamine-6P Synthase. DOI:10.1074/jbc.m511689200. PMID:16339762.
(2002), Nat Prod Rep, 19, 60-69. From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase. DOI:10.1039/b103713g.
Milewski S (2002), Biochim Biophys Acta, 1597, 173-192. Glucosamine-6-phosphate synthase—the multi-facets enzyme. DOI:10.1016/s0167-4838(02)00318-7. PMID:12044898.
Teplyakov A et al. (2001), J Mol Biol, 313, 1093-1102. Channeling of ammonia in glucosamine-6-phosphate synthase. DOI:10.1006/jmbi.2001.5094. PMID:11700065.
Oinonen C et al. (2000), Protein Sci, 9, 2329-2337. Structural comparison of Ntn-hydrolases. DOI:10.1110/ps.9.12.2329. PMID:11206054.
Bearne SL et al. (2000), J Biol Chem, 275, 135-140. Inhibition of Escherichia coliGlucosamine-6-phosphate Synthase by Reactive Intermediate Analogues: THE ROLE OF THE 2-AMINO FUNCTION IN CATALYSIS. DOI:10.1074/jbc.275.1.135.
Teplyakov A et al. (1999), Protein Sci, 8, 596-602. The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase. DOI:10.1110/ps.8.3.596. PMID:10091662.
Teplyakov A et al. (1998), Structure, 6, 1047-1055. 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. PMID:9739095.
Isupov MN et al. (1996), Structure, 4, 801-810. Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 å crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase. DOI:10.1016/s0969-2126(96)00087-1. PMID:8805567.
Bearne SL et al. (1995), Biochemistry, 34, 11515-11520. Glutamate gamma-semialdehyde as a natural transition state analogue inhibitor of Escherichia coli glucosamine-6-phosphate synthase. PMID:7547881.
Obmolova G et al. (1994), J Mol Biol, 242, 703-705. Crystallization and Preliminary X-ray Analysis of the Two Domains of Glucosamine-6-phosphate Synthase from Escherichia coli. DOI:10.1006/jmbi.1994.1619. PMID:7932726.

Step 1. The N-terminus of Cys1 deprotonates water, which then deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction.

Residue	Roles
Asn98A	hydrogen bond donor, electrostatic stabiliser
Gly99A (main-N)	hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term)	hydrogen bond acceptor
Arg26A (main-C)	hydrogen bond acceptor, activator
Trp74A (main-N)	hydrogen bond donor, electrostatic stabiliser
Cys1A	proton donor, nucleophile
Cys1A (N-term)	proton acceptor

Residue	Roles
His504A(AA)	hydrogen bond donor
Glu481A	hydrogen bond acceptor, electrostatic stabiliser
Lys485A	hydrogen bond donor, electrostatic stabiliser
Lys603A	hydrogen bond donor
His504A(AA)	proton donor

Glutamine-fructose-6-phosphate transaminase (isomerizing)

Reference Protein and Structure

Enzyme Reaction (EC:2.6.1.16)

Enzyme Mechanism

Introduction

Catalytic Residues Roles

Chemical Components

References

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Catalytic Residues Roles

Chemical Components

Contributors