DB code: S00200

RLCP classification 6.10.445000.115 : Double-bonded atom exchange
5.111.850000.6300 : Elimination
6.20.29000.6000 : Double-bonded atom exchange
CATH domain 3.20.20.70 : TIM Barrel Catalytic domain
E.C. 4.1.3.3
CSA 1fdy
M-CSA 1fdy
MACiE

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 S00235 S00239 S00240 S00243 S00244 S00199 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
P0A6L4 N-acetylneuraminate lyase
EC 4.1.3.3
N-acetylneuraminic acid aldolase
N-acetylneuraminate pyruvate-lyase
Sialic acid lyase
Sialate lyase
Sialic acid aldolase
NALase
NP_417692.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_491409.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
PF00701 (DHDPS)
[Graphical View]
P44539 N-acetylneuraminate lyase
EC 4.1.3.3
N-acetylneuraminic acid aldolase
N-acetylneuraminate pyruvate-lyase
Sialic acid lyase
Sialate lyase
Sialic acid aldolase
NP_438311.1 (Protein)
NC_000907.1 (DNA/RNA sequence)
PF00701 (DHDPS)
[Graphical View]

KEGG enzyme name
N-acetylneuraminate lyase
N-acetylneuraminic acid aldolase
acetylneuraminate lyase
sialic aldolase
sialic acid aldolase
sialate lyase
N-acetylneuraminic aldolase
neuraminic aldolase
N-acetylneuraminate aldolase
neuraminic acid aldolase
N-acetylneuraminic acid aldolase
neuraminate aldolase
N-acetylneuraminic lyase
N-acetylneuraminic acid lyase
NPL
NALase
NANA lyase
acetylneuraminate pyruvate-lyase
N-acetylneuraminate pyruvate-lyase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P0A6L4 NANA_ECOLI N-acetylneuraminate = N-acetyl-D-mannosamine + pyruvate. Homotetramer. Cytoplasm.
P44539 NANA_HAEIN N-acetylneuraminate = N-acetyl-D-mannosamine + pyruvate. Homotetramer. Cytoplasm.

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

Compound table
Substrates Products Intermediates
KEGG-id C00270 C00645 C00022
E.C. (Neu5Ac)
(pyruvate)
Compound N-Acetylneuraminate N-Acetyl-D-mannosamine Pyruvate Schiff-base with substrate Schiff-base with product
Type amide group,carbohydrate,carboxyl group amide group,carbohydrate carbohydrate,carboxyl group
ChEBI 17012
 17012
 		63153
 63153
 		32816
 32816
PubChem 439197
 439197
 		439281
 439281
 		1060
 1060
1f5zA Unbound Unbound Unbound Unbound Unbound
1f5zB Unbound Unbound Unbound Unbound Unbound
1f5zC Unbound Unbound Unbound Unbound Unbound
1f5zD Unbound Unbound Unbound Unbound Unbound
1f6kA Unbound Unbound Unbound Unbound Unbound
1f6kC Unbound Unbound Unbound Unbound Unbound
1f6pA Unbound Unbound Unbound Unbound Unbound
1f6pB Unbound Unbound Unbound Unbound Unbound
1f6pC Unbound Unbound Unbound Unbound Unbound
1f6pD Unbound Unbound Unbound Unbound Unbound
1f73A Analogue:HMN Unbound Unbound Unbound Unbound
1f73B Analogue:HMN Unbound Unbound Unbound Unbound
1f73C Analogue:HMN Unbound Unbound Unbound Unbound
1f73D Analogue:HMN Unbound Unbound Unbound Unbound
1f74A Analogue:NAY Unbound Unbound Unbound Unbound
1f74C Analogue:NAY Unbound Unbound Unbound Unbound
1f7bA Unbound Unbound Unbound Intermediate-analogue:NAU Unbound
1f7bC Unbound Unbound Unbound Intermediate-analogue:NAV Unbound
1fdyA Unbound Unbound Unbound Unbound Intermediate-analogue:3PY
1fdyB Unbound Unbound Unbound Unbound Intermediate-analogue:3PY
1fdyC Unbound Unbound Unbound Unbound Intermediate-analogue:3PY
1fdyD Unbound Unbound Unbound Unbound Intermediate-analogue:3PY
1fdzA Unbound Unbound Bound:PYR Unbound Intermediate-bound:PYR
1fdzB Unbound Unbound Bound:PYR Unbound Intermediate-bound:PYR
1fdzC Unbound Unbound Bound:PYR Unbound Intermediate-bound:PYR
1fdzD Unbound Unbound Bound:PYR Unbound Intermediate-bound:PYR
1nal1 Unbound Unbound Unbound Unbound Unbound
1nal2 Unbound Unbound Unbound Unbound Unbound
1nal3 Unbound Unbound Unbound Unbound Unbound
1nal4 Unbound Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;P0A6L4, P44539

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1f5zA TYR 136;LYS 164 GLY 188
1f5zB TYR 136;LYS 164 GLY 188
1f5zC TYR 136;LYS 164 GLY 188
1f5zD TYR 136;LYS 164 GLY 188
1f6kA TYR 136;LYS 164 GLY 188
1f6kC TYR 136;LYS 164 GLY 188
1f6pA TYR 136;LYS 164 GLY 188
1f6pB TYR 136;LYS 164 GLY 188
1f6pC TYR 136;LYS 164 GLY 188
1f6pD TYR 136;LYS 164 GLY 188
1f73A TYR 136;LYS 164 GLY 188
1f73B TYR 136;LYS 164 GLY 188
1f73C TYR 136;LYS 164 GLY 188
1f73D TYR 136;LYS 164 GLY 188
1f74A TYR 136;LYS 164 GLY 188
1f74C TYR 136;LYS 164 GLY 188
1f7bA TYR 136;LYS 164 GLY 188
1f7bC TYR 136;LYS 164 GLY 188
1fdyA TYR 137;LYS 165 GLY 189
1fdyB TYR 137;LYS 165 GLY 189
1fdyC TYR 137;LYS 165 GLY 189
1fdyD TYR 137;LYS 165 GLY 189
1fdzA TYR 137;LYS 165 GLY 189
1fdzB TYR 137;LYS 165 GLY 189
1fdzC TYR 137;LYS 165 GLY 189
1fdzD TYR 137;LYS 165 GLY 189
1nal1 TYR 137;LYS 165 GLY 189
1nal2 TYR 137;LYS 165 GLY 189
1nal3 TYR 137;LYS 165 GLY 189
1nal4 TYR 137;LYS 165 GLY 189

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[6]
[9]
Fig.1(a), Fig.6, p.394-395
[11]
[13]
Fig.9, p.416-418 9

References
[1]
Resource
Comments
Medline ID
PubMed ID 6301475
Journal Biochem Biophys Res Commun
Year 1983
Volume 111
Pages 668-74
Authors Deijl CM, Vliegenthart JF
Title Configuration of substrate and products of N-acetylneuraminate pyruvate-lyase from Clostridium perfringens.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 3024643
Journal Biochem Int
Year 1986
Volume 13
Pages 493-500
Authors Kolisis FN, Hervagault JF
Title Theoretical and experimental study on the competition of NANA-aldolase and cytidine-5'-monophosphosialate-synthase for their common substrate N-acetylneuraminic acid.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 2896604
Journal FEBS Lett
Year 1988
Volume 232
Pages 145-7
Authors Gross HJ, Brossmer R
Title Inhibition of N-acetylneuraminate lyase by N-acetyl-4-oxo-D-neuraminic acid.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 2896358
Journal Prog Clin Biol Res
Year 1988
Volume 259
Pages 413-20
Authors Kolisis FN, Evangelopoulos AE
Title Studies on metabolic regulation and estimation of sialic acids by enzyme immobilization techniques.
Related PDB
Related UniProtKB
[5]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.2 ANGSTROMS).
Medline ID 94363237
PubMed ID 8081752
Journal Structure
Year 1994
Volume 2
Pages 361-9
Authors Izard T, Lawrence MC, Malby RL, Lilley GG, Colman PM
Title The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli.
Related PDB 1nal
Related UniProtKB P0A6L4
[6]
Resource
Comments
Medline ID
PubMed ID
Journal J Org Chem
Year 1995
Volume 60
Pages 3663-70
Authors Fitz W, Schwark JR, Wong CH
Title Aldotetroses and C(3)-modified aldohexoses as substrates for N-acetylneuraminic acid aldolase: a model for the explanation of the normal and the inversed stereoselectivity.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 8581891
Journal Carbohydr Res
Year 1996
Volume 280
Pages 101-10
Authors Murakami M, Ikeda K, Achiwa K
Title Chemoenzymatic synthesis of neuraminic acid analogs structurally varied at C-5 and C-9 as potential inhibitors of the sialidase from influenza virus.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 8704962
Journal Microbiology
Year 1996
Volume 142
Pages 1221-30
Authors Homer KA, Kelley S, Hawkes J, Beighton D, Grootveld MC
Title Metabolism of glycoprotein-derived sialic acid and N-acetylglucosamine by Streptococcus oralis.
Related PDB
Related UniProtKB
[9]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.45 ANGSTROMS) OF COMPLEXES WITH HYDROXYPYRUVATE AND PYRUVATE.
Medline ID 97199395
PubMed ID 9047371
Journal J Mol Biol
Year 1997
Volume 266
Pages 381-99
Authors Lawrence MC, Barbosa JA, Smith BJ, Hall NE, Pilling PA, Ooi HC, Marcuccio SM
Title Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase.
Related PDB 1fdy 1fdz
Related UniProtKB P0A6L4
[10]
Resource
Comments
Medline ID
PubMed ID 9535696
Journal Protein Expr Purif
Year 1998
Volume 12
Pages 295-304
Authors Lilley GG, Barbosa JA, Pearce LA
Title Expression in Escherichia coli of the putative N-acetylneuraminate lyase gene (nanA) from Haemophilus influenzae: overproduction, purification, and crystallization.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 11674166
Journal J Org Chem
Year 1999
Volume 64
Pages 945-49
Authors Smith BJ, Lawrence MC, Barbosa JA
Title Substrate-Assisted Catalysis in Sialic Acid Aldolase.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 10732983
Journal Bioorg Med Chem
Year 2000
Volume 8
Pages 657-64
Authors Kiefelt MJ, Wilson JC, Bennett S, Gredley M, von Itzstein M
Title Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase.
Related PDB
Related UniProtKB
[13]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.6 ANGSTROMS) OF NATIVE PROTEIN AND COMPLEXES WITH SIALIC ACID ALDITOL; 4-DEOXY-SIALIC ACID AND 4-OXO-SIALIC ACID.
Medline ID 20487594
PubMed ID 11031117
Journal J Mol Biol
Year 2000
Volume 303
Pages 405-21
Authors Barbosa JA, Smith BJ, DeGori R, Ooi HC, Marcuccio SM, Campi EM, Jackson WR, Brossmer R, Sommer M, Lawrence MC
Title Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.
Related PDB 1f5z 1f6k 1f6p 1f73 1f74 1f7b
Related UniProtKB P44539
[14]
Resource
Comments
Medline ID
PubMed ID 11434370
Journal Carbohydr Res
Year 2001
Volume 332
Pages 133-9
Authors Kok GB, Campbell M, Mackey BL, von Itzstein M
Title Synthesis of C-3 nitrogen-containing derivatives of N-acetyl-alpha,beta-D-mannosamine as substrates for N-acetylneuraminic acid aldolase.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 11504613
Journal Trends Biochem Sci
Year 2001
Volume 26
Pages 468-9
Authors Baardsnes J, Davies PL
Title Sialic acid synthase: the origin of fish type III antifreeze protein?
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 12083785
Journal Biochem Biophys Res Commun
Year 2002
Volume 295
Pages 167-73
Authors Hwang TS, Hung CH, Teo CF, Chen GT, Chang LS, Chen SF, Chen YJ, Lin CH
Title Structural characterization of Escherichia coli sialic acid synthase.
Related PDB
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID 12453637
Journal Biochimie
Year 2002
Volume 84
Pages 655-60
Authors Bulai T, Bratosin D, Artenie V, Montreuil J
Title Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID 12150863
Journal Bioorg Med Chem
Year 2002
Volume 10
Pages 3175-85
Authors Humphrey AJ, Fremann C, Critchley P, Malykh Y, Schauer R, Bugg TD
Title Biological properties of N-acyl and N-haloacetyl neuraminic acids: processing by enzymes of sialic acid metabolism, and interaction with influenza virus.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID 12711733
Journal Proc Natl Acad Sci U S A
Year 2003
Volume 100
Pages 5694-9
Authors Joerger AC, Mayer S, Fersht AR
Title Mimicking natural evolution in vitro: an N-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity.
Related PDB
Related UniProtKB

Comments
(B) Schiff-base formation.
(B1) Lys164 makes a nucleophilic attack on the C2 carbonyl carbon, forming carbinolamine cation. Here, a proton transfer from the nitrogen of Lys164 to the carbonyl oxygen occurs.
This enzyme catalyzes the following reactions (according to the literature [13]):
(A) Ring-opening through eliminative double-bond formation.
(B2) Further transfer of a proton from the nitrogen to the hydroxyl oxgen of the carbinolamine cation gives the oxonium intermediate. (These proton transfers might be assisted by Tyr136 as acid-base.)
(B3) The lone pair of the Lys amine attacks on the C2 carbon, leading to the dehydration of the oxonium, which results in the formation of Schiff-base intermediate (or immonium intermediate) and double-bond between the nitrogen and the C2 atom.
(B4) Gly188 oxygen atom might stabilize the Schiff-base, indirectly by interacting with the O4 oxygen atom.
(C) Eliminative double-bond formation.
(C1) Substrate carboxylate acts as a general base to deprotonate the O4 hydroxylg group, through the sidechain of Tyr136.
(C2) The deprotonation of the hydroxyl group leads to the bond cleavage between the C4 and C3 atoms, resulting in the formation of the O4 carbonyl oxygen and the enamine intermediate bound to Lys164. This step releases a product, N-acetylmannosamine.
(D) Isomerization (change in the position of double-bond; tautomerization).
(D1) Substrate carboxylate acts as a general acid to protonate the C3 methylene carbon atom, through the sidechain of Tyr136, leading to the formation of imine intermediate from the enamine.
(E) Schiff-base deformation.
(E1) Water makes a nucleophilic attack on the C2 carbon, forming the oxonium intermediate.
(E2) A proton transfer from the oxonium group to the Lys164 nitrogen leads to the formation of carbinolamine cation.
(E3) The lone pair of the hydroxyl oxygen attacks on the C2 carbon, leading to the elimination of Lys164, which results in the formation of another product, pyruvate.
The ring-closure of N-acetylmannosamine occurs spontaneously (see [13]).

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