M-CSA Mechanism and Catalytic Site Atlas

Methylaspartate ammonia-lyase

Methylasparate ammonium lyase (MAL) is involved in the methylaspartate cycle and catalyses the formation of the alpha,beta-unsaturated bond by the reversible anti elimination of ammonia from L-threo-beta-methylaspartate (L-threo-(2S,3S)-3-methylaspartate) to give mesaconate. It can also use L-erythro-beta-methylaspartate (L-erythro-(2S,3R)-3-methylaspartate), L-aspartate, fumarate and ethylfumarate as substrates. It will also catalyse the stereo- and regioselective addition of ammonia to several derivatives of mesaconic acid to give a limited number of homochiral substituted aspartic acids. The structure of MAL is similar to that of members of the enolase superfamily, which all catalyse removal of a proton alpha to a carboxylic acid as the first step in their mechanism.

Reference Protein and Structure

Sequence: Q05514 (4.3.1.2) (Sequence Homologues) (PDB Homologues)
Biological species: Clostridium tetanomorphum (Bacteria)
PDB: 1kcz - Crystal Structure of beta-methylaspartase from Clostridium tetanomorphum. Mg-complex. (1.9 Å)
Catalytic CATH Domains: 3.20.20.120 (see all for 1kcz)
Cofactors: Magnesium(2+) (1)

Click To Show Structure

Enzyme Reaction (EC:4.3.1.2)

threo-3-methyl-L-aspartate(1-)

CHEBI:58724

→

mesaconate(2-)

CHEBI:36986

ammonium

CHEBI:28938

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: 3-methylaspartase, Beta-methylaspartase, L-threo-3-methylaspartate ammonia-lyase,

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

Summary
Step 1
Step 2
Step 3
Products
All Steps

Introduction

Lys 331 acts as a general base to remove the 3-proton from 3-methyl aspartate to generate an enolic intermediate. An Mg(II) cation and His 194 provide positive charges to stabilise the accumulation of negative charge on the substrate carboxyl group during the formation of this intermediate. Collapse of the enolate leads to elimination of ammonia in an E1cB mechanism.

Catalytic Residues Roles

UniProt	PDB* (1kcz)
His194	His194A	Provides a positive charge to stabilise the negative charge that accumulates in the enolate intermediate following abstraction of the 3-proton.	electrostatic stabiliser
Lys331	Lys331A	Acts as a general base to remove the 3-proton and generate an enolic intermediate.	proton acceptor, electrostatic stabiliser, proton donor
Gln172	Gln172A	Binds the amine group of the substrate. Provides electrostatic stabilisation during the elimination step of the reaction to this group.	electrostatic stabiliser
Asp238, Glu273, Asp307	Asp238A, Glu273A, Asp307A	Forms the magnesium binding site.	metal ligand
Gln329	Gln329A	Involved in the stabilisation of the enolate anion intermediate.	electrostatic stabiliser

*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, assisted keto-enol tautomerisation, unimolecular elimination by the conjugate base, inferred reaction step

References

de Villiers M et al. (2012), ACS Chem Biol, 7, 1618-1628. Catalytic Mechanisms and Biocatalytic Applications of Aspartate and Methylaspartate Ammonia Lyases. DOI:10.1021/cb3002792. PMID:22834890.
Raj H et al. (2013), FEBS Open Bio, 3, 285-290. The roles of active site residues in the catalytic mechanism of methylaspartate ammonia-lyase. DOI:10.1016/j.fob.2013.07.002. PMID:23905011.
Raj H et al. (2009), Chembiochem, 10, 2236-2245. Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based Mutagenesis. DOI:10.1002/cbic.200900311. PMID:19670200.
Asuncion M et al. (2002), J Biol Chem, 277, 8306-8311. The Structure of 3-Methylaspartase from Clostridium tetanomorphum Functions via the Common Enolase Chemical Step. DOI:10.1074/jbc.m111180200. PMID:11748244.
Levy CW et al. (2002), Structure, 10, 105-113. Insights into Enzyme Evolution Revealed by the Structure of Methylaspartate Ammonia Lyase. DOI:10.1016/s0969-2126(01)00696-7. PMID:11796115.
Goda SK et al. (1992), Biochemistry, 31, 10747-10756. Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding beta-methylaspartase and characterization of the recombinant protein. PMID:1420191.

Step 1. Lys331 abstracts the alpha proton from the substrate.

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Catalytic Residues Roles

Residue	Roles
His194A	electrostatic stabiliser
Gln172A	electrostatic stabiliser
Gln329A	electrostatic stabiliser
Asp238A	metal ligand
Glu273A	metal ligand
Asp307A	metal ligand
Lys331A	proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation

Step 2. The negatively charged intermediate collapses, eliminating ammonia.

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Catalytic Residues Roles

Residue	Roles
Asp238A	metal ligand
Glu273A	metal ligand
Asp307A	metal ligand
Gln172A	electrostatic stabiliser
Gln329A	electrostatic stabiliser
His194A	electrostatic stabiliser
Lys331A	electrostatic stabiliser