L-aspartate oxidase

 

L-aspartate oxidase (LASPO) is a flavo enzyme from Escherichia coli. It is unusual as it can use both oxygen and fumarate as electron acceptors for FAD oxidation, which means it is both aerobic and anaerobic. LASPO is an FAD dependent enzyme which catalyses the oxidation of L-aspartate into iminoaspartate. This is the first step in the bacterial synthesis of NAD+, and is a useful target for drug design, as this pathway is not present in mammals.

 

Reference Protein and Structure

Sequence
P10902 UniProt (1.4.3.16) IPR005288 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1knp - E. coli L-aspartate oxidase: mutant R386L in complex with succinate (2.6 Å) PDBe PDBsum 1knp
Catalytic CATH Domains
3.50.50.60 CATHdb 3.90.700.10 CATHdb (see all for 1knp)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.4.3.16)

L-aspartate(1-)
CHEBI:29991ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
iminoaspartate(1-)
CHEBI:77875ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
Alternative enzyme names: NadB, Laspo, AO, LASPO,

Enzyme Mechanism

Introduction

Arg 290 acts as a base by deprotonating C3 of L-aspartate. Arg 386 and His 351 stabilise the negative charge on the O4 oxygen atoms. FAD then accepts a hydride from C2 of L-aspartate (and most likely then accepts a proton from the solvent to form FADH2.)

If fumarate is used as the electron acceptor FADH- donates a hydride to the C2 atom of fumarate. The negative charge is localised on the O4 atoms which are stabilised by Arg386 and His 351. The C4 carbonyl is reformed, and Arg 290 donates a proton to the C3 atom, forming succinate.

Alternative molecular oxygen may re-oxidize FADH2, forming hydrogen peroxide.

Catalytic Residues Roles

UniProt PDB* (1knp)
Arg290 Arg290A Acts as a base and deprotonates the C3 atom of L-aspartate. Acts as an acid in its protonated form, and donates a proton to the C3 atom of fumerate. proton acceptor, proton donor
Glu121, His244, Arg386, His351 Glu121A, His244A, Leu386A, His351A Acts to stabilise the negatively charged intermediate. electrostatic stabiliser, steric role
*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, hydride transfer, aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, bimolecular nucleophilic addition, native state of cofactor regenerated

References

  1. Bossi RT et al. (2002), Biochemistry, 41, 3018-3024. Structure of FAD-Boundl-Aspartate Oxidase:  Insight into Substrate Specificity and Catalysis†,‡. DOI:10.1021/bi015939r. PMID:11863440.
  2. Tedeschi G et al. (2010), Biochimie, 92, 1335-1342. On the catalytic role of the active site residue E121 of E. coli L-aspartate oxidase. DOI:10.1016/j.biochi.2010.06.015. PMID:20600565.
  3. Messner KR et al. (2002), J Biol Chem, 277, 42563-42571. Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase. DOI:10.1074/jbc.M204958200. PMID:12200425.

Step 1. Arg290 acts as a base to deprotonate C3 of the substrate. Glu121 orientates the substrate in a productive binding mode and promotes proton abstraction. FAD then accepts a hydride from C2, resulting in the enamine tautomer of the product and FADH-. The enamine will consequently tautomerise to form the imine product.

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Catalytic Residues Roles

Residue Roles
His351A electrostatic stabiliser
Leu386A electrostatic stabiliser
Glu121A electrostatic stabiliser
His244A electrostatic stabiliser
Glu121A steric role
Arg290A proton acceptor

Chemical Components

proton transfer, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used

Step 2. Fumarate reduction involved hydride transfer from FADH- N5 to fumarate C2, coupled with proton donation by Arg290. FAD and the active site residues are regenerated. Alternatively, molecular oxygen may be used to oxidise FADH- instead of fumarate (not shown here).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg290A proton donor

Chemical Components

hydride transfer, proton transfer, ingold: bimolecular nucleophilic addition, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. Arg290 acts as a base to deprotonate C3 of the substrate. Glu121 orientates the substrate in a productive binding mode and promotes proton abstraction. FAD then accepts a hydride from C2, resulting in the enamine tautomer of the product and FADH-. The enamine will consequently tautomerise to form the imine product.

Step 2. Fumarate reduction involved hydride transfer from FADH- N5 to fumarate C2, coupled with proton donation by Arg290. FAD and the active site residues are regenerated. Alternatively, molecular oxygen may be used to oxidise FADH- instead of fumarate (not shown here).

Products.

Introduction

Kinetic isotope effect experimental data argue against the succinate dehydrogenase-like mechanism for aspartate oxidation and for a concerted mechanism. The mechanism proposal shown here involves Glu121 may act as a base to deprotonate the amino group of L-aspartate, followed by hydride transfer from C2 to N5 of FAD. This forms the product and FADH-. It is also possible for the mechanism to involve deprotonation of C2 of L-aspartate followed by hydride transfer from N1 to FAD. FADH- may be reoxidised using fumarate, or may abstract a proton from the solvent to form FADH2 and be reoxidised by molecular oxygen.

Catalytic Residues Roles

UniProt PDB* (1knp)
Glu121 Glu121A Glu121 may act as a base to deprotonate L-aspartate in the first step of the mechanism. proton acceptor, electrostatic stabiliser
Arg290 Arg290A Arg290 may act as a base to deprotonate L-aspartate in the first step of the mechanism. Arg290 is involved in the fumarate reduction reaction in which it acts as a general acid. electrostatic stabiliser, proton donor
His244, Arg386, His351 His244A, Leu386A, His351A The residues act as electrostatic stabilisers. 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

hydride transfer, proton transfer, aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, cofactor used, bimolecular nucleophilic addition, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Chow C et al. (2017), Biochemistry, 56, 4044-4052. Mechanistic Characterization of Escherichia coli l-Aspartate Oxidase from Kinetic Isotope Effects. DOI:10.1021/acs.biochem.7b00307. PMID:28700220.
  2. Bossi RT et al. (2002), Biochemistry, 41, 3018-3024. Structure of FAD-Boundl-Aspartate Oxidase:  Insight into Substrate Specificity and Catalysis†,‡. DOI:10.1021/bi015939r. PMID:11863440.

Step 1. Glu121 binds to the protonated α-amino group of L-aspartate and acts as a base to perform the initial deprotonation of the amine. In a concerted reaction, the C2 hydride of L-aspartate is transferred to N5 of FAD. This results in the product and FADH-.

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Catalytic Residues Roles

Residue Roles
His244A electrostatic stabiliser
Arg290A electrostatic stabiliser
His351A electrostatic stabiliser
Glu121A proton acceptor

Chemical Components

hydride transfer, proton transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, cofactor used

Step 2. FADH- is reoxidised by fumarate. A hydride is transferred from N5 of FADH- while fumarate accepts a proton from a general acid donor, most likely to be Arg290. Molecular oxygen can also be used to regenerate FAD from FADH2, forming hydrogen peroxide in the process.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu121A electrostatic stabiliser
His244A electrostatic stabiliser
His351A electrostatic stabiliser
Arg290A proton donor

Chemical Components

hydride transfer, proton transfer, ingold: bimolecular nucleophilic addition, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu121 binds to the protonated α-amino group of L-aspartate and acts as a base to perform the initial deprotonation of the amine. In a concerted reaction, the C2 hydride of L-aspartate is transferred to N5 of FAD. This results in the product and FADH-.

Step 2. FADH- is reoxidised by fumarate. A hydride is transferred from N5 of FADH- while fumarate accepts a proton from a general acid donor, most likely to be Arg290. Molecular oxygen can also be used to regenerate FAD from FADH2, forming hydrogen peroxide in the process.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Amelia Brasnett