N-acetylneuraminate lyase

 

The N-acetylneuraminate lyase (NAL) enzymes are a subfamily of the (beta/alpha)8 enzymes. They all share a common catalytic step but catalyse reactions in different biological pathways. The formation of a Schiff base between a strictly conserved lysine residue and the C2 carbon of the common keto-acid moiety of the substrate is a common feature of the group, with the NAL enzymes catalysing the aldol cleavage of N-acetylneuraminate to form N-acetylmannosine and pyruvate via the distinctive Schiff base intermediate.

 

Reference Protein and Structure

Sequence
P0A6L4 UniProt (4.1.3.3) IPR005264 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1fdy - N-ACETYLNEURAMINATE LYASE IN COMPLEX WITH HYDROXYPYRUVATE (2.45 Å) PDBe PDBsum 1fdy
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1fdy)
Click To Show Structure

Enzyme Reaction (EC:4.1.3.3)

N-acetylneuraminate
CHEBI:35418ChEBI
→
aldehydo-N-acetyl-D-mannosamine
CHEBI:17122ChEBI
+
pyruvate
CHEBI:15361ChEBI
Alternative enzyme names: N-acetylneuraminate aldolase, N-acetylneuraminic acid aldolase, N-acetylneuraminic acid lyase, N-acetylneuraminic aldolase, N-acetylneuraminic lyase, NALase, NANA lyase, NPL, Acetylneuraminate lyase, Acetylneuraminate pyruvate-lyase, Neuraminate aldolase, Neuraminic acid aldolase, Neuraminic aldolase, Sialate lyase, Sialic acid aldolase, Sialic aldolase, N-acetylneuraminate pyruvate-lyase,

Enzyme Mechanism

Introduction

The initial step is thought to be ring opening of the substrate alpha anomer. The conserved Lys165 then acts as nucleophile to the carbonyl group with concomitant proton transfer from the Lys165 nitrogen to the alcohol group of the tetrahedral intermediate. A second proton transfer results in a cationic oxonium group. The attacking nitrogen lone pair then kicks out a water molecule forming the substrate-enzyme Schiff base intermediate. A tyrosine group mediates the proton abstraction from the hydroxyl at the 4 position by the carboxylate group, leading to the formation of an enamine. Protonation at the methylene carbon then converts the enamine into an imine. Subsequent ring closure of N-acetylmannosine is spontaneous. The collapse of the enzyme-substrate intermediate is brought about by a hydrolytic water molecule which leads to the liberation of Lys165 and pyruvate.

Catalytic Residues Roles

UniProt PDB* (1fdy)
Lys165 Lys165A Forms a Schiff-base intermediate with the substrate. Also involved in ring opening. covalently attached, nucleofuge, nucleophile, proton acceptor, electron pair acceptor, electron pair donor
Tyr137 Tyr137A Involved in proton transfer during cleavage. proton relay, 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

decyclisation, proton transfer, overall reactant used, intermediate formation, bimolecular nucleophilic addition, intramolecular elimination, schiff base formed, bimolecular elimination, overall product formed, proton relay, cyclisation, tautomerisation (not keto-enol), intermediate terminated, native state of enzyme regenerated

References

  1. Lawrence MC et al. (1997), J Mol Biol, 266, 381-399. Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase. DOI:10.1006/jmbi.1996.0769. PMID:9047371.
  2. Daniels AD et al. (2014), ACS Chem Biol, 9, 1025-1032. Reaction mechanism of N-acetylneuraminic acid lyase revealed by a combination of crystallography, QM/MM simulation, and mutagenesis. DOI:10.1021/cb500067z. PMID:24521460.
  3. Barbosa JA et al. (2000), J Mol Biol, 303, 405-421. Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme. DOI:10.1006/jmbi.2000.4138. PMID:11031117.

Step 1. The reaction begins with the ring opening. Which is promoted by Lys165.

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Catalytic Residues Roles

Residue Roles
Lys165A proton acceptor

Chemical Components

decyclisation, proton transfer

Step 2. Lys165 performs a nucleophilic attack upon C2 forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Lys165A covalently attached, nucleophile

Chemical Components

overall reactant used, intermediate formation, ingold: bimolecular nucleophilic addition, proton transfer

Step 3. Water is eliminated from this intermediate and an imine is formed.

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Catalytic Residues Roles

Residue Roles
Lys165A covalently attached, electron pair donor

Chemical Components

ingold: intramolecular elimination, schiff base formed

Step 4. The bond between C3 and C4 is broken releasing the linear form of the product aldehydo-N-acetyl-D-mannosamine and an enamine intermediate is formed. This is assisted by Tyr143 which acts as a proton relay.

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Catalytic Residues Roles

Residue Roles
Lys165A covalently attached
Tyr137A proton relay, proton donor
Lys165A electron pair acceptor
Tyr137A proton acceptor

Chemical Components

ingold: bimolecular elimination, overall product formed, proton relay

Step 5. Aldehydo-N-acetyl-D-mannosamine is converted to its cyclic form. The enamine tautomerizes forming a new imine intermediate.

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Catalytic Residues Roles

Residue Roles
Lys165A covalently attached, electron pair donor

Chemical Components

proton transfer, cyclisation, tautomerisation (not keto-enol), schiff base formed

Step 6. A hydrolytic water performs a nucleophilic attack upon the imine carbon forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Lys165A covalently attached, electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition

Step 7. Lys165 is eliminated from the intermediate and the other product pyruvate is formed.

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Catalytic Residues Roles

Residue Roles
Lys165A nucleofuge

Chemical Components

ingold: bimolecular elimination, proton transfer, overall product formed, intermediate terminated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The reaction begins with the ring opening. Which is promoted by Lys165.

Step 2. Lys165 performs a nucleophilic attack upon C2 forming a tetrahedral intermediate.

Step 3. Water is eliminated from this intermediate and an imine is formed.

Step 4. The bond between C3 and C4 is broken releasing the linear form of the product aldehydo-N-acetyl-D-mannosamine and an enamine intermediate is formed. This is assisted by Tyr143 which acts as a proton relay.

Step 5. Aldehydo-N-acetyl-D-mannosamine is converted to its cyclic form. The enamine tautomerizes forming a new imine intermediate.

Step 6. A hydrolytic water performs a nucleophilic attack upon the imine carbon forming a tetrahedral intermediate.

Step 7. Lys165 is eliminated from the intermediate and the other product pyruvate is formed.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday, James Willey, Yordanos Abeje