Lanthanide-dependent methanol dehydrogenase

 

Lanthanide-dependent methanol dehydrogenase is a widespread enzyme used by methylotrophic bacteria to oxidise methanol for carbon and energy. The enzyme requires early lanthanide (Ln) metals in the 3+ oxidation state for catalytic activity.

Across the series (La-Lu), catalytic activity of Ln-MDH decreases; Eu-MDH shows significantly lower catalytic activity than Ce-MDH, and Yb-MDH shows no catalytic activity. This is attributed to the lanthanide contraction effect whereby the ionic radius of Ln ions decreases as atomic number increases. In turn metal-ligand bond lengths decreases and Lewis acidity increases. Despite this, however, we observe a decrease in activity across the series.

Nearly all strains of Methylobacterium have at least two types of MDHs, such as MxaFI, a Ca2+-dependent MDH, and XoxF, an Ln-dependent MDH. In contrast to Ca2+-dependent MDH (EC 1.1.2.7), Ln-dependent MDH shows little activity with Ca2+.

 

Reference Protein and Structure

Sequence
I0JWN7 UniProt (1.1.2.7, 1.1.2.8) IPR017512 (Sequence Homologues) (PDB Homologues)
Biological species
Methylacidiphilum fumariolicum SolV (Bacteria) Uniprot
PDB
6fkw - Europium-containing methanol dehydrogenase (1.4 Å) PDBe PDBsum 6fkw
Catalytic CATH Domains
(see all for 6fkw)
Cofactors
Europium (iii) ion (1)
Click To Show Structure

Enzyme Reaction (EC:1.1.2.10)

methanol
CHEBI:17790ChEBI
+
pyrroloquinoline quinone
CHEBI:18315ChEBI
→
formaldehyde
CHEBI:16842ChEBI
+
pyrroloquinoline quinol
CHEBI:18356ChEBI
Alternative enzyme names: La(3+)-dependent MDH, Ce(3+)-induced methanol dehydrogenase, Cerium dependent MDH,

Enzyme Mechanism

Introduction

A possible mechanism for oxidation of methanol to formaldehyde by MDH is an addition-elimination-protonation reaction. In this reaction, a lanthanide ion is directly bound to the pyrroloquinolinequinone cofactor (PQQ). The enzyme shows highest activity with early Ln, but a later-Ln (Eu) is shown here.

In the first step (addition), the oxygen atom of the methanol substrate attacks the carbonyl carbon of PQQ. Simultaneously, the hydroxyl proton is donated to Asp299 which is acting as a general base. The next step (elimination) releases the formaldehyde product as a result of protonation of PQQ by the substrate, forming PQQH. A second protonation, this time by Asp299, forms PQQH2 and regenerates the enzyme active site.

Catalytic Residues Roles

UniProt PDB* (6fkw)
Asn290, Asp335, Glu206 Asn256A, Asp301A, Glu172A Asn256, Asp301 and Glu172 coordinate the Ln ion via their negatively charged side chains. metal ligand
Asp333 Asp299A Asp299 is coordinated to the Ln centre, and acts as a general acid/base. metal ligand, 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

bimolecular nucleophilic addition, proton transfer, intermediate formation, cofactor used, decoordination from a metal ion, overall reactant used, rate-determining step, overall product formed, intermediate collapse, unimolecular elimination by the conjugate base, native state of enzyme regenerated, native state of cofactor is not regenerated

References

  1. Prejanò M et al. (2020), Chemistry, 26, 11334-11339. How Lanthanide Ions Affect the Addition-Elimination Step of Methanol Dehydrogenases. DOI:10.1002/chem.202001855. PMID:32369635.
  2. Knasin AL et al. (2021), Methods Enzymol, 650, 19-55. Synthetic modeling of the structure and function of the rare-earth dependent methanol dehydrogenase cofactor. DOI:10.1016/bs.mie.2021.01.037. PMID:33867022.
  3. Pastawan V et al. (2020), 8, 186-198. Biological Function of Lanthanide in Plant-Symbiotic Bacteria: Lanthanide-Dependent Methanol Oxidation System. DOI:10.7831/ras.8.0_186.
  4. McSkimming A et al. (2018), J Am Chem Soc, 140, 1223-1226. Functional Synthetic Model for the Lanthanide-Dependent Quinoid Alcohol Dehydrogenase Active Site. DOI:10.1021/jacs.7b12318. PMID:29286650.
  5. Prejanò M et al. (2017), Chemistry, 23, 8652-8657. How Can Methanol Dehydrogenase from Methylacidiphilum fumariolicum Work with the Alien CeIII Ion in the Active Center? A Theoretical Study. DOI:10.1002/chem.201700381. PMID:28488399.

Step 1. The oxygen of the methanol substrate attacks the carbonyl carbon of the PQQ redox cofactor and simultaneously donates its proton to Asp299, which acts as a general base.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn256A metal ligand
Asp301A metal ligand
Glu172A metal ligand
Asp299A metal ligand
Asp299A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, cofactor used, decoordination from a metal ion, overall reactant used, rate-determining step

Step 2. The formaldehyde product is released as a result of protonation of PQQ by the substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn256A metal ligand
Asp301A metal ligand
Glu172A metal ligand
Asp299A metal ligand

Chemical Components

overall product formed, intermediate collapse, cofactor used, proton transfer, ingold: unimolecular elimination by the conjugate base

Step 3. A second protonation of the cofactor forms PQQH2 and deprotonated Asp299.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp299A proton donor
Asn256A metal ligand
Asp301A metal ligand
Glu172A metal ligand
Asp299A metal ligand

Chemical Components

proton transfer, native state of enzyme regenerated, native state of cofactor is not regenerated
Download: Image, Marvin File

Step 1. The oxygen of the methanol substrate attacks the carbonyl carbon of the PQQ redox cofactor and simultaneously donates its proton to Asp299, which acts as a general base.

Step 2. The formaldehyde product is released as a result of protonation of PQQ by the substrate.

Step 3. A second protonation of the cofactor forms PQQH2 and deprotonated Asp299.

Products.

Introduction

The first step of this mechanism, supported by computational studies, is a hydride transfer from the methanol substrate to the carbonyl carbon of PQQ. Asp299 acts as a general base, accepting a proton from the hydroxyl group of methanol. Asp299 then donates a proton to PQQ. Keto-enol tautomerisation forms PQQH2.

Catalytic Residues Roles

UniProt PDB* (6fkw)
Asn290, Asp335, Glu206 Asn256A, Asp301A, Glu172A Asn256, Asp301 and Glu172 coordinate the Ln ion via their negatively charged side chains. metal ligand
Asp333 Asp299A Asp299 is coordinated to the Ln centre, and acts as a general acid/base. metal ligand, 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

overall reactant used, decoordination from a metal ion, cofactor used, intermediate formation, proton transfer, hydride transfer, rate-determining step, native state of enzyme regenerated, assisted keto-enol tautomerisation, native state of cofactor is not regenerated, overall product formed

References

  1. McSkimming A et al. (2018), J Am Chem Soc, 140, 1223-1226. Functional Synthetic Model for the Lanthanide-Dependent Quinoid Alcohol Dehydrogenase Active Site. DOI:10.1021/jacs.7b12318. PMID:29286650.
  2. Knasin AL et al. (2021), Methods Enzymol, 650, 19-55. Synthetic modeling of the structure and function of the rare-earth dependent methanol dehydrogenase cofactor. DOI:10.1016/bs.mie.2021.01.037. PMID:33867022.
  3. Pastawan V et al. (2020), 8, 186-198. Biological Function of Lanthanide in Plant-Symbiotic Bacteria: Lanthanide-Dependent Methanol Oxidation System. DOI:10.7831/ras.8.0_186.

Step 1. Hydride transfer from the methanol substrate to the carbonyl carbon of PQQ. Asp299 acts as a general base, accepting a proton from the hydroxyl group of methanol.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp299A metal ligand
Glu172A metal ligand
Asp301A metal ligand
Asn256A metal ligand
Asp299A proton acceptor

Chemical Components

overall reactant used, decoordination from a metal ion, cofactor used, intermediate formation, proton transfer, hydride transfer, rate-determining step

Step 2. Proton transfer from Asp299 to PQQ.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp299A proton donor
Asn256A metal ligand
Asp301A metal ligand
Glu172A metal ligand
Asp299A metal ligand

Chemical Components

proton transfer, native state of enzyme regenerated

Step 3. Keto-enol tautomerisation forms PQQH2.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn256A metal ligand
Asp301A metal ligand
Glu172A metal ligand
Asp299A metal ligand

Chemical Components

assisted keto-enol tautomerisation, native state of cofactor is not regenerated, overall product formed
Download: Image, Marvin File

Step 1. Hydride transfer from the methanol substrate to the carbonyl carbon of PQQ. Asp299 acts as a general base, accepting a proton from the hydroxyl group of methanol.

Step 2. Proton transfer from Asp299 to PQQ.

Step 3. Keto-enol tautomerisation forms PQQH2.

Products.

Introduction

A retro-ene mechanism has been considered for the methanol reduction. As in the addition-elimination mechanism, in the first step, the oxygen atom of the methanol substrate attacks the carbonyl carbon of PQQ and the hydroxyl proton is donated to Asp299. In this case, PQQ is first protonated by Asp299 and then by the substrate, which releases the formaldehyde product. Computational studies comparing both the addition-elimination and retro-ene mechanisms rule out this mechanism.

Catalytic Residues Roles

UniProt PDB* (6fkw)
Asn290, Asp335, Glu206 Asn256A, Asp301A, Glu172A Asn256, Asp301 and Glu172 coordinate the Ln ion via their negatively charged side chains. metal ligand
Asp333 Asp299A Asp299 is coordinated to the Ln centre, and acts as a general acid/base. metal ligand, 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

rate-determining step, overall reactant used, decoordination from a metal ion, cofactor used, intermediate formation, proton transfer, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

References

  1. Prejanò M et al. (2017), Chemistry, 23, 8652-8657. How Can Methanol Dehydrogenase from Methylacidiphilum fumariolicum Work with the Alien CeIII Ion in the Active Center? A Theoretical Study. DOI:10.1002/chem.201700381. PMID:28488399.
  2. Pastawan V et al. (2020), 8, 186-198. Biological Function of Lanthanide in Plant-Symbiotic Bacteria: Lanthanide-Dependent Methanol Oxidation System. DOI:10.7831/ras.8.0_186.
  3. Prejanò M et al. (2020), Chemistry, 26, 11334-11339. How Lanthanide Ions Affect the Addition-Elimination Step of Methanol Dehydrogenases. DOI:10.1002/chem.202001855. PMID:32369635.

Step 1. The oxygen of the methanol substrate attacks the carbonyl carbon of the PQQ redox cofactor and simultaneously donates its proton to Asp299, which acts as a general base.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp299A metal ligand
Glu172A metal ligand
Asp301A metal ligand
Asn256A metal ligand
Asp299A proton acceptor

Chemical Components

rate-determining step, overall reactant used, decoordination from a metal ion, cofactor used, intermediate formation, proton transfer, ingold: bimolecular nucleophilic addition

Step 2. Protonation of PQQ by Asp299.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp299A metal ligand
Glu172A metal ligand
Asp301A metal ligand
Asn256A metal ligand
Asp299A proton donor

Chemical Components

proton transfer, cofactor used

Step 3. The formaldehyde product is released as a result of protonation of PQQ by the substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp299A metal ligand
Glu172A metal ligand
Asp301A metal ligand
Asn256A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, cofactor used, intermediate collapse, overall product formed
Download: Image, Marvin File

Step 1. The oxygen of the methanol substrate attacks the carbonyl carbon of the PQQ redox cofactor and simultaneously donates its proton to Asp299, which acts as a general base.

Step 2. Protonation of PQQ by Asp299.

Step 3. The formaldehyde product is released as a result of protonation of PQQ by the substrate.

Products.

Contributors

Noa Marson, Antonio Ribeiro