EzCatDB: S00200
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DB codeS00200
RLCP classification6.10.445000.115 : Double-bonded atom exchange
5.111.850000.6300 : Elimination
6.20.29000.6000 : Double-bonded atom exchange
CATH domainDomain 13.20.20.70 : TIM BarrelCatalytic domain
E.C.4.1.3.3
CSA1fdy

CATH domainRelated DB codes (homologues)
3.20.20.70 : TIM BarrelS00215,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

Enzyme Name
UniProtKBKEGG

P0A6L4P44539
Protein nameN-acetylneuraminate lyaseN-acetylneuraminate lyaseN-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
SynonymsEC 4.1.3.3
N-acetylneuraminic acid aldolase
N-acetylneuraminate pyruvate-lyase
Sialic acid lyase
Sialate lyase
Sialic acid aldolase
NALase
EC 4.1.3.3
N-acetylneuraminic acid aldolase
N-acetylneuraminate pyruvate-lyase
Sialic acid lyase
Sialate lyase
Sialic acid aldolase
RefSeqNP_417692.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_491409.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
NP_438311.1 (Protein)
NC_000907.1 (DNA/RNA sequence)
PfamPF00701 (DHDPS)
[Graphical view]
PF00701 (DHDPS)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00530Aminosugars metabolism

UniProtKB:Accession NumberP0A6L4P44539
Entry nameNANA_ECOLINANA_HAEIN
ActivityN-acetylneuraminate = N-acetyl-D-mannosamine + pyruvate.N-acetylneuraminate = N-acetyl-D-mannosamine + pyruvate.
SubunitHomotetramer.Homotetramer.
Subcellular locationCytoplasm.Cytoplasm.
Cofactor


Compound table: links to PDB-related databases & PoSSuM

SubstratesProductsintermediates
KEGG-idC00270C00645C00022

CompoundN-AcetylneuraminateN-Acetyl-D-mannosaminePyruvateSchiff-base with substrate Schiff-base with product
Typeamide group,carbohydrate,carboxyl groupamide group,carbohydratecarbohydrate,carboxyl group

ChEBI17012
63153
32816


PubChem439197
439281
1060


             
1f5zAUnboundUnboundUnboundUnboundUnbound
1f5zBUnboundUnboundUnboundUnboundUnbound
1f5zCUnboundUnboundUnboundUnboundUnbound
1f5zDUnboundUnboundUnboundUnboundUnbound
1f6kAUnboundUnboundUnboundUnboundUnbound
1f6kCUnboundUnboundUnboundUnboundUnbound
1f6pAUnboundUnboundUnboundUnboundUnbound
1f6pBUnboundUnboundUnboundUnboundUnbound
1f6pCUnboundUnboundUnboundUnboundUnbound
1f6pDUnboundUnboundUnboundUnboundUnbound
1f73AAnalogue:HMNUnboundUnboundUnboundUnbound
1f73BAnalogue:HMNUnboundUnboundUnboundUnbound
1f73CAnalogue:HMNUnboundUnboundUnboundUnbound
1f73DAnalogue:HMNUnboundUnboundUnboundUnbound
1f74AAnalogue:NAYUnboundUnboundUnboundUnbound
1f74CAnalogue:NAYUnboundUnboundUnboundUnbound
1f7bAUnboundUnboundUnboundIntermediate-analogue:NAUUnbound
1f7bCUnboundUnboundUnboundIntermediate-analogue:NAVUnbound
1fdyAUnboundUnboundUnboundUnboundIntermediate-analogue:3PY
1fdyBUnboundUnboundUnboundUnboundIntermediate-analogue:3PY
1fdyCUnboundUnboundUnboundUnboundIntermediate-analogue:3PY
1fdyDUnboundUnboundUnboundUnboundIntermediate-analogue:3PY
1fdzAUnboundUnboundBound:PYRUnboundIntermediate-bound:PYR
1fdzBUnboundUnboundBound:PYRUnboundIntermediate-bound:PYR
1fdzCUnboundUnboundBound:PYRUnboundIntermediate-bound:PYR
1fdzDUnboundUnboundBound:PYRUnboundIntermediate-bound:PYR
1nal1UnboundUnboundUnboundUnboundUnbound
1nal2UnboundUnboundUnboundUnboundUnbound
1nal3UnboundUnboundUnboundUnboundUnbound
1nal4UnboundUnboundUnboundUnboundUnbound

Active-site residues
resource
Swiss-prot;P0A6L4, P44539
pdbCatalytic residuesMain-chain involved in catalysis
          
1f5zATYR 136;LYS 164
GLY 188
1f5zBTYR 136;LYS 164
GLY 188
1f5zCTYR 136;LYS 164
GLY 188
1f5zDTYR 136;LYS 164
GLY 188
1f6kATYR 136;LYS 164
GLY 188
1f6kCTYR 136;LYS 164
GLY 188
1f6pATYR 136;LYS 164
GLY 188
1f6pBTYR 136;LYS 164
GLY 188
1f6pCTYR 136;LYS 164
GLY 188
1f6pDTYR 136;LYS 164
GLY 188
1f73ATYR 136;LYS 164
GLY 188
1f73BTYR 136;LYS 164
GLY 188
1f73CTYR 136;LYS 164
GLY 188
1f73DTYR 136;LYS 164
GLY 188
1f74ATYR 136;LYS 164
GLY 188
1f74CTYR 136;LYS 164
GLY 188
1f7bATYR 136;LYS 164
GLY 188
1f7bCTYR 136;LYS 164
GLY 188
1fdyATYR 137;LYS 165
GLY 189
1fdyBTYR 137;LYS 165
GLY 189
1fdyCTYR 137;LYS 165
GLY 189
1fdyDTYR 137;LYS 165
GLY 189
1fdzATYR 137;LYS 165
GLY 189
1fdzBTYR 137;LYS 165
GLY 189
1fdzCTYR 137;LYS 165
GLY 189
1fdzDTYR 137;LYS 165
GLY 189
1nal1TYR 137;LYS 165
GLY 189
1nal2TYR 137;LYS 165
GLY 189
1nal3TYR 137;LYS 165
GLY 189
1nal4TYR 137;LYS 165
GLY 189

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[6]

[9]Fig.1(a), Fig.6, p.394-395
[11]

[13]Fig.9, p.416-4189

references
[1]
PubMed ID6301475
JournalBiochem Biophys Res Commun
Year1983
Volume111
Pages668-74
AuthorsDeijl CM, Vliegenthart JF
TitleConfiguration of substrate and products of N-acetylneuraminate pyruvate-lyase from Clostridium perfringens.
[2]
PubMed ID3024643
JournalBiochem Int
Year1986
Volume13
Pages493-500
AuthorsKolisis FN, Hervagault JF
TitleTheoretical and experimental study on the competition of NANA-aldolase and cytidine-5'-monophosphosialate-synthase for their common substrate N-acetylneuraminic acid.
[3]
PubMed ID2896604
JournalFEBS Lett
Year1988
Volume232
Pages145-7
AuthorsGross HJ, Brossmer R
TitleInhibition of N-acetylneuraminate lyase by N-acetyl-4-oxo-D-neuraminic acid.
[4]
PubMed ID2896358
JournalProg Clin Biol Res
Year1988
Volume259
Pages413-20
AuthorsKolisis FN, Evangelopoulos AE
TitleStudies on metabolic regulation and estimation of sialic acids by enzyme immobilization techniques.
[5]
CommentsX-RAY CRYSTALLOGRAPHY (2.2 ANGSTROMS).
Medline ID94363237
PubMed ID8081752
JournalStructure
Year1994
Volume2
Pages361-9
AuthorsIzard T, Lawrence MC, Malby RL, Lilley GG, Colman PM
TitleThe three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli.
Related PDB1nal
Related UniProtKBP0A6L4
[6]
JournalJ Org Chem
Year1995
Volume60
Pages3663-70
AuthorsFitz W, Schwark JR, Wong CH
TitleAldotetroses and C(3)-modified aldohexoses as substrates for N-acetylneuraminic acid aldolase: a model for the explanation of the normal and the inversed stereoselectivity.
[7]
PubMed ID8581891
JournalCarbohydr Res
Year1996
Volume280
Pages101-10
AuthorsMurakami M, Ikeda K, Achiwa K
TitleChemoenzymatic synthesis of neuraminic acid analogs structurally varied at C-5 and C-9 as potential inhibitors of the sialidase from influenza virus.
[8]
PubMed ID8704962
JournalMicrobiology
Year1996
Volume142
Pages1221-30
AuthorsHomer KA, Kelley S, Hawkes J, Beighton D, Grootveld MC
TitleMetabolism of glycoprotein-derived sialic acid and N-acetylglucosamine by Streptococcus oralis.
[9]
CommentsX-RAY CRYSTALLOGRAPHY (2.45 ANGSTROMS) OF COMPLEXES WITH HYDROXYPYRUVATE AND PYRUVATE.
Medline ID97199395
PubMed ID9047371
JournalJ Mol Biol
Year1997
Volume266
Pages381-99
AuthorsLawrence MC, Barbosa JA, Smith BJ, Hall NE, Pilling PA, Ooi HC, Marcuccio SM
TitleStructure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase.
Related PDB1fdy,1fdz
Related UniProtKBP0A6L4
[10]
PubMed ID9535696
JournalProtein Expr Purif
Year1998
Volume12
Pages295-304
AuthorsLilley GG, Barbosa JA, Pearce LA
TitleExpression in Escherichia coli of the putative N-acetylneuraminate lyase gene (nanA) from Haemophilus influenzae: overproduction, purification, and crystallization.
[11]
PubMed ID11674166
JournalJ Org Chem
Year1999
Volume64
Pages945-49
AuthorsSmith BJ, Lawrence MC, Barbosa JA
TitleSubstrate-Assisted Catalysis in Sialic Acid Aldolase.
[12]
PubMed ID10732983
JournalBioorg Med Chem
Year2000
Volume8
Pages657-64
AuthorsKiefelt MJ, Wilson JC, Bennett S, Gredley M, von Itzstein M
TitleSynthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase.
[13]
CommentsX-RAY CRYSTALLOGRAPHY (1.6 ANGSTROMS) OF NATIVE PROTEIN AND COMPLEXES WITH SIALIC ACID ALDITOL; 4-DEOXY-SIALIC ACID AND 4-OXO-SIALIC ACID.
Medline ID20487594
PubMed ID11031117
JournalJ Mol Biol
Year2000
Volume303
Pages405-21
AuthorsBarbosa JA, Smith BJ, DeGori R, Ooi HC, Marcuccio SM, Campi EM, Jackson WR, Brossmer R, Sommer M, Lawrence MC
TitleActive site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.
Related PDB1f5z,1f6k,1f6p,1f73,1f74,1f7b
Related UniProtKBP44539
[14]
PubMed ID11434370
JournalCarbohydr Res
Year2001
Volume332
Pages133-9
AuthorsKok GB, Campbell M, Mackey BL, von Itzstein M
TitleSynthesis of C-3 nitrogen-containing derivatives of N-acetyl-alpha,beta-D-mannosamine as substrates for N-acetylneuraminic acid aldolase.
[15]
PubMed ID11504613
JournalTrends Biochem Sci
Year2001
Volume26
Pages468-9
AuthorsBaardsnes J, Davies PL
TitleSialic acid synthase: the origin of fish type III antifreeze protein?
[16]
PubMed ID12083785
JournalBiochem Biophys Res Commun
Year2002
Volume295
Pages167-73
AuthorsHwang TS, Hung CH, Teo CF, Chen GT, Chang LS, Chen SF, Chen YJ, Lin CH
TitleStructural characterization of Escherichia coli sialic acid synthase.
[17]
PubMed ID12453637
JournalBiochimie
Year2002
Volume84
Pages655-60
AuthorsBulai T, Bratosin D, Artenie V, Montreuil J
TitleCharacterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes.
[18]
PubMed ID12150863
JournalBioorg Med Chem
Year2002
Volume10
Pages3175-85
AuthorsHumphrey AJ, Fremann C, Critchley P, Malykh Y, Schauer R, Bugg TD
TitleBiological properties of N-acyl and N-haloacetyl neuraminic acids: processing by enzymes of sialic acid metabolism, and interaction with influenza virus.
[19]
PubMed ID12711733
JournalProc Natl Acad Sci U S A
Year2003
Volume100
Pages5694-9
AuthorsJoerger AC, Mayer S, Fersht AR
TitleMimicking natural evolution in vitro: an N-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity.

comments
This enzyme catalyzes the following reactions (according to the literature [13]):
(A) Ring-opening through eliminative double-bond formation.
(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.
(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]).

createdupdated
2004-05-262009-02-26
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 2008)
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)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
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