DB code: S00235

RLCP classification	8.131.708000.451 : Isomerization
	4.151.766500.451 : Addition
	5.151.2325000.451 : Elimination
	8.113.915300.451 : Isomerization
CATH domain	3.20.20.70 : TIM Barrel	Catalytic domain
E.C.	4.1.2.13
CSA	1b57
M-CSA	1b57
MACiE	M0052

CATH domain	Related DB codes (homologues)
3.20.20.70 : TIM Barrel	S00215 S00217 S00218 S00219 S00532 S00198 S00220 S00745 S00537 S00538 S00539 S00826 S00841 S00239 S00240 S00243 S00244 S00199 S00200 S00201 S00221 S00222 S00847 S00224 S00225 S00226 D00014 D00029 M00141 T00015 T00239 D00664 D00665 D00804 D00863 T00089

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	RefSeq	Pfam
P0AB71	Fructose-bisphosphate aldolase class 2	FBP aldolase FBPA EC 4.1.2.13 Fructose-bisphosphate aldolase class II Fructose-1,6-bisphosphate aldolase	NP_417400.1 (Protein) NC_000913.2 (DNA/RNA sequence) YP_491125.1 (Protein) NC_007779.1 (DNA/RNA sequence)	PF01116 (F_bP_aldolase) [Graphical View]

KEGG enzyme name
fructose-bisphosphate aldolase aldolase fructose-1,6-bisphosphate triosephosphate-lyase fructose diphosphate aldolase diphosphofructose aldolase fructose 1,6-diphosphate aldolase ketose 1-phosphate aldolase phosphofructoaldolase zymohexase fructoaldolase fructose 1-phosphate aldolase fructose 1-monophosphate aldolase 1,6-Diphosphofructose aldolase SMALDO D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase

KEGG enzyme name

fructose-bisphosphate aldolase
aldolase
fructose-1,6-bisphosphate triosephosphate-lyase
fructose diphosphate aldolase
diphosphofructose aldolase
fructose 1,6-diphosphate aldolase
ketose 1-phosphate aldolase
phosphofructoaldolase
zymohexase
fructoaldolase
fructose 1-phosphate aldolase
fructose 1-monophosphate aldolase
1,6-Diphosphofructose aldolase
SMALDO
D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P0AB71	ALF_ECOLI	D-fructose 1,6-bisphosphate = glycerone phosphate + D-glyceraldehyde 3-phosphate.	Homodimer.		Zinc.

KEGG Pathways
Map code	Pathways	E.C.
MAP00010	Glycolysis / Gluconeogenesis
MAP00030	Pentose phosphate pathway
MAP00051	Fructose and mannose metabolism
MAP00710	Carbon fixation in photosynthetic organisms

Compound table
	Cofactors		Substrates	Products		Intermediates
KEGG-id	C00038	C99999	C00354	C00111	C00118
E.C.
Compound	Zinc	Monovalent metal	D-Fructose 1,6-bisphosphate	Glycerone phosphate	D-Glyceraldehyde 3-phosphate
Type	heavy metal	univalent metal (Na+, K+)	carbohydrate,phosphate group/phosphate ion	carbohydrate,phosphate group/phosphate ion	carbohydrate,phosphate group/phosphate ion
ChEBI	29105 29105		37736 37736	16108 16108	29052 29052
PubChem	32051 32051		172313 172313	668 668	439168 439168

1b57A	Bound:_ZN_360	Bound:_NA	Unbound	Analogue:PGH	Unbound
1b57B	Bound:_ZN_360	Bound:_NA	Unbound	Analogue:PGH	Unbound
1dosA	Bound:_ZN	Unbound	Unbound	Unbound	Unbound
1dosB	Bound:_ZN	Unbound	Unbound	Unbound	Unbound
1zenA	Bound:2x_ZN	Unbound	Unbound	Unbound	Unbound
1gynA	Analogue:_CD_401	Unbound	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
Swiss-prot;P0AB71 & literature [8], [10] & [12]

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

1b57A	ASP 109;GLU 182;ASN 286	HIS 110;GLU 174;HIS 226;HIS 264(Catalytic zinc);VAL 225;GLY 227;GLY 265;SER 267(Monovalent metal)
1b57B	ASP 109;GLU 182;ASN 286	HIS 110;GLU 174;HIS 226;HIS 264(Catalytic zinc);VAL 225;GLY 227;GLY 265;SER 267(Monovalent metal)
1dosA	ASP 109;GLU 182;ASN 286	HIS 110;GLU 174;HIS 226;HIS 264(Catalytic zinc);VAL 225;GLY 227;GLY 265;SER 267(Monovalent metal)
1dosB	ASP 109;GLU 182;ASN 286	HIS 110;GLU 174;HIS 226;HIS 264(Catalytic zinc);VAL 225;GLY 227;GLY 265;SER 267(Monovalent metal)
1zenA	ASP 109;;ASN 286	HIS 110;GLU 174;HIS 226;HIS 264(Catalytic zinc);VAL 225;GLY 227;GLY 265;SER 267(Monovalent metal)			invisible 177-193
1gynA	ASP 109;;ASN 286	HIS 110;GLU 174;;HIS 264(Catalytic zinc);VAL 225;;GLY 265;SER 267(Monovalent metal)			invisible 177-193, 226-243

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[1]	Fig.9, p.1203-1204	2
[5]	p.250-251
[8]	p.858-859
[9]	Fig.5, p.157
[10]	p.1307-1310
[12]	Fig.6, p.389-391	5
[13]	Fig.3, p.852
[14]	Fig.1	5
[18]	Fig.2, p.137-138	6

References
[1]
Resource
Comments
Medline ID
PubMed ID	4324205
Journal	Biochemistry
Year	1971
Volume	10
Pages	1191-204
Authors	Mildvan AS, Kobes RD, Rutter WJ
Title	Magnetic resonance studies of the role of the divalent cation in the mechanism of yeast aldolase.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID	4607364
Journal	Biochemistry
Year	1974
Volume	13
Pages	4371-5
Authors	Heron EJ, Caprioli RM
Title	18O studies of the mechanisms of yeast and muscle aldolases.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID	1613797
Journal	J Mol Biol
Year	1992
Volume	225
Pages	1137-41
Authors	Naismith JH, Ferrara JD, Bailey S, Marshall K, Dauter Z, Wilson KS, Habash J, Harrop SJ, Berry AJ, Hunter WN
Title	Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase.
Related PDB
Related UniProtKB
[4]
Resource
Comments	ZINC-LIGANDS, AND MUTAGENESIS.
Medline ID	93170474
PubMed ID	8436219
Journal	FEBS Lett
Year	1993
Volume	318
Pages	11-6
Authors	Berry A, Marshall KE
Title	Identification of zinc-binding ligands in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.
Related PDB
Related UniProtKB	P0AB71
[5]
Resource
Comments
Medline ID
PubMed ID	7872790
Journal	Arch Biochem Biophys
Year	1995
Volume	317
Pages	244-52
Authors	Szwergold BS, Ugurbil K, Brown TR
Title	Properties of fructose-1,6-bisphosphate aldolase from Escherichia coli: an NMR analysis.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	8898912
Journal	Eur J Biochem
Year	1996
Volume	241
Pages	243-8
Authors	De Montigny C, Sygusch J
Title	Functional characterization of an extreme thermophilic class II fructose-1,6-bisphosphate aldolase.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	9174952
Journal	J Mol Recognit
Year	1996
Volume	9
Pages	652-7
Authors	Nicholls IA, Matsui J, Krook M, Mosbach K
Title	Some recent developments in the preparation of novel recognition systems: a recognition site for the selective catalysis of an aldol condensation using molecular imprinting and specific affinity motifs for alpha-chymotrypsin using a phage display peptide library.
Related PDB
Related UniProtKB
[8]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.67 ANGSTROMS).
Medline ID	96433074
PubMed ID	8836102
Journal	Nat Struct Biol
Year	1996
Volume	3
Pages	856-62
Authors	Blom NS, Tetreault S, Coulombe R, Sygusch J
Title	Novel active site in Escherichia coli fructose 1,6-bisphosphate aldolase.
Related PDB	1dos
Related UniProtKB	P0AB71
[9]
Resource
Comments
Medline ID
PubMed ID	8771208
Journal	Protein Sci
Year	1996
Volume	5
Pages	154-61
Authors	Qamar S, Marsh K, Berry A
Title	Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.
Related PDB
Related UniProtKB
[10]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS).
Medline ID	97094986
PubMed ID	8939754
Journal	Structure
Year	1996
Volume	4
Pages	1303-15
Authors	Cooper SJ, Leonard GA, McSweeney SM, Thompson AW, Naismith JH, Qamar S, Plater A, Berry A, Hunter WN
Title	The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold.
Related PDB	1zen
Related UniProtKB	P0AB71
[11]
Resource
Comments
Medline ID
PubMed ID	9548961
Journal	Biochemistry
Year	1998
Volume	37
Pages	5746-54
Authors	Johnson AE, Tanner ME
Title	Epimerization via carbon-carbon bond cleavage. L-ribulose-5-phosphate 4-epimerase as a masked class II aldolase.
Related PDB
Related UniProtKB
[12]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID	99182425
PubMed ID	10080900
Journal	J Mol Biol
Year	1999
Volume	287
Pages	383-94
Authors	Hall DR, Leonard GA, Reed CD, Watt CI, Berry A, Hunter WN
Title	The crystal structure of Escherichia coli class II fructose-1, 6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity.
Related PDB	1b57
Related UniProtKB	P0AB71
[13]
Resource
Comments
Medline ID
PubMed ID	9878448
Journal	J Mol Biol
Year	1999
Volume	285
Pages	843-55
Authors	Plater AR, Zgiby SM, Thomson GJ, Qamar S, Wharton CW, Berry A
Title	Conserved residues in the mechanism of the E. coli Class II FBP-aldolase.
Related PDB
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID	10712619
Journal	Eur J Biochem
Year	2000
Volume	267
Pages	1858-68
Authors	Zgiby SM, Thomson GJ, Qamar S, Berry A
Title	Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID	11173490
Journal	Acta Crystallogr D Biol Crystallogr
Year	2001
Volume	57
Pages	310-3
Authors	Sauve V, Sygusch J
Title	Crystallization and preliminary X-ray analysis of native and selenomethionine fructose-1,6-bisphosphate aldolase from Thermus aquaticus.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID	11371431
Journal	Biophys J
Year	2001
Volume	80
Pages	2527-35
Authors	Ouporov IV, Knull HR, Huber A, Thomasson KA
Title	Brownian dynamics simulations of aldolase binding glyceraldehyde 3-phosphate dehydrogenase and the possibility of substrate channeling.
Related PDB
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID	11237691
Journal	Protein Expr Purif
Year	2001
Volume	21
Pages	293-302
Authors	Sauve V, Sygusch J
Title	Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID	11779234
Journal	J Mol Biol
Year	2002
Volume	315
Pages	131-40
Authors	Zgiby S, Plater AR, Bates MA, Thomson GJ, Berry A
Title	A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID	12595741
Journal	Acta Crystallogr D Biol Crystallogr
Year	2003
Volume	59
Pages	611-4
Authors	Hall DR, Kemp LE, Leonard GA, Marshall K, Berry A, Hunter WN
Title	The organization of divalent cations in the active site of cadmium Escherichia coli fructose-1,6-bisphosphate aldolase.
Related PDB
Related UniProtKB

Comments
Although Glu174 is bound to the catalytic zinc ion, it moves away from the zinc ion to allow the substrate/product (Glycerone phosphate; GP) moiety to coordinate the ion during the catalysis (see [10]). Here, zinc ion functions as a Lewis acid electron sink, and polarizes the carbonyl bond of GP, for the elimination or addition reaction. A monovalent cation is required for substrate binding, although it is not involved in catalysis. According to the literature [18], although Glu182 is distant from the active site in the absence of substrate, it would be brought into a position to act as a general base on binding of substrates. This enzyme catalyzes two reverse reactions, which are composed of two reactions respectively (see [12]): (#1) the aldol condensation of a ketose, Glycerone phosphate (GP) and an aldose, glyceraldehyde 3-phosphate (G3P) to form the acyclic form of fructose 1,6-bisphospahte (FBP); that is addition of GP to the double-bond (carbonyl group) of G3P. (In Gluconeogenesis pathway) (A) Isomerization: (B) Addition of GP to the double-bond (carbonyl group) of G3P: (#2) the clevage of FBP into GP and G3P; that is elimination of GP from FBP to form a new double-bond in another product, G3P. (In glycolysis pathway) (C) Elimination of GP: (D) Isomerization: These catalytic reactions proceeds as follows (see [12] & [18]): (A) Isomerization: (A1) Carbonyl group and hydroxyl group of GP are bound to the catalytic zinc ion, replacing Glu174. Here, the zinc ion polarizes the carbonyl bond of the ketose substrate, GP, which increases the acidity of the hydroxymethylene (C1) hydrogen atoms, facilitating the next reaction, deprotonation. (A2) Glu182 acts as a general base to deprotonate the C1 atom, leading to the formation of ene-diolate (carbanion) intermediate. This intermediate is stabilized by Asn286. (B) Addition of the ene-diolate intermediate to the double-bond (carbonyl group) of G3P (B1) The second substrate (aldehyde), G3P, which is electrophilic, binds to the active site, to interact with the nucleophilic ene-diolate intermediate. (The two planes of the ene-diolate nucleophile and the carbonyl acceptor must be nearly parallel for the C-C bond formation.) Here, Asp109 polarizes the addition site, the C1 carbonyl group of G3P (as a modulator). (B2) The C1 atom of the ene-diolate intermediate makes a nucleophilic attack on the C1 carbonyl carbon of G3P, forming C-C bond. Here, Asp109 protonate the carbonyl oxygen of G3P, leading to formation of the hydroxyl group. At the same time, a carbonyl group is formed at the C2 carbon of the product, FBP. On the other hand, the reverse reaction (C) proceeds as follows: (C) Elimination of GP from FBP to form a new double-bond in G3P (C1) The carbonyl oxygen (O2) and C3 hydroxyl group of FBP are bound to the catalytic zinc ion. This zinc ion polarizes the carbonyl bond of FBP. (C2) Asp109 acts as a general base to deprotonate the C4 hydroxyl group of FBP (deprotonation site of leaving group), leading to the C-C bond cleavage. This cleavage gives the ene-diolate intermediate, which is bound to the catalytic zinc ion, and a leaving product, G3P. The ene-diolate intermediate is stabilized by Asn286. (C3) G3P is released from the active site. (D) Isomerization: (D1) Glu182 acts as a general acid to protonate the C1 sp2 carbon of the intermediate, to form the carbonyl group at the C2 carbon.

Comments

Although Glu174 is bound to the catalytic zinc ion, it moves away from the zinc ion to allow the substrate/product (Glycerone phosphate; GP) moiety to coordinate the ion during the catalysis (see [10]). Here, zinc ion functions as a Lewis acid electron sink, and polarizes the carbonyl bond of GP, for the elimination or addition reaction.
A monovalent cation is required for substrate binding, although it is not involved in catalysis.
According to the literature [18], although Glu182 is distant from the active site in the absence of substrate, it would be brought into a position to act as a general base on binding of substrates.
This enzyme catalyzes two reverse reactions, which are composed of two reactions respectively (see [12]):
(#1) the aldol condensation of a ketose, Glycerone phosphate (GP) and an aldose, glyceraldehyde 3-phosphate (G3P) to form the acyclic form of fructose 1,6-bisphospahte (FBP); that is addition of GP to the double-bond (carbonyl group) of G3P. (In Gluconeogenesis pathway)
(A) Isomerization:
(B) Addition of GP to the double-bond (carbonyl group) of G3P:
(#2) the clevage of FBP into GP and G3P; that is elimination of GP from FBP to form a new double-bond in another product, G3P. (In glycolysis pathway)
(C) Elimination of GP:
(D) Isomerization:
These catalytic reactions proceeds as follows (see [12] & [18]):
(A) Isomerization:
(A1) Carbonyl group and hydroxyl group of GP are bound to the catalytic zinc ion, replacing Glu174. Here, the zinc ion polarizes the carbonyl bond of the ketose substrate, GP, which increases the acidity of the hydroxymethylene (C1) hydrogen atoms, facilitating the next reaction, deprotonation.
(A2) Glu182 acts as a general base to deprotonate the C1 atom, leading to the formation of ene-diolate (carbanion) intermediate. This intermediate is stabilized by Asn286.
(B) Addition of the ene-diolate intermediate to the double-bond (carbonyl group) of G3P
(B1) The second substrate (aldehyde), G3P, which is electrophilic, binds to the active site, to interact with the nucleophilic ene-diolate intermediate. (The two planes of the ene-diolate nucleophile and the carbonyl acceptor must be nearly parallel for the C-C bond formation.) Here, Asp109 polarizes the addition site, the C1 carbonyl group of G3P (as a modulator).
(B2) The C1 atom of the ene-diolate intermediate makes a nucleophilic attack on the C1 carbonyl carbon of G3P, forming C-C bond. Here, Asp109 protonate the carbonyl oxygen of G3P, leading to formation of the hydroxyl group. At the same time, a carbonyl group is formed at the C2 carbon of the product, FBP.
On the other hand, the reverse reaction (C) proceeds as follows:
(C) Elimination of GP from FBP to form a new double-bond in G3P
(C1) The carbonyl oxygen (O2) and C3 hydroxyl group of FBP are bound to the catalytic zinc ion. This zinc ion polarizes the carbonyl bond of FBP.
(C2) Asp109 acts as a general base to deprotonate the C4 hydroxyl group of FBP (deprotonation site of leaving group), leading to the C-C bond cleavage. This cleavage gives the ene-diolate intermediate, which is bound to the catalytic zinc ion, and a leaving product, G3P. The ene-diolate intermediate is stabilized by Asn286.
(C3) G3P is released from the active site.
(D) Isomerization:
(D1) Glu182 acts as a general acid to protonate the C1 sp2 carbon of the intermediate, to form the carbonyl group at the C2 carbon.

Created	Updated
2004-05-26	2009-02-26