Recombinant Photorhabdus luminescens subsp. laumondii 2,3-dihydroxyphenylpropionate/2,3-dihydroxicinnamic acid 1,2-dioxygenase (mhpB)

Definition and Biological Context

MhpB is a non-heme iron-dependent extradiol dioxygenase that catalyzes the oxidative ring cleavage of catecholic intermediates, such as 2,3-dihydroxyphenylpropionate (DHPP) and 2,3-dihydroxicinnamic acid (DHCI), during the degradation of aromatic compounds like 3-phenylpropionic acid (PP) and cinnamic acid (CI) . While native to E. coli, recombinant expression in other organisms (including potential P. luminescens systems) could leverage its catalytic versatility for biodegradation applications.

2.1. CryoEM Structure and Subunit Arrangement

The 2.59 Å resolution cryoEM structure of MhpB (PDB ID: 9kti) reveals a decameric assembly with a catalytic chamber positioned at the side of the structure. Key features include:

• Iron-binding site: A non-heme Fe(II) coordinated by conserved residues (e.g., His-115, His-179, and Asp-114).

• Substrate-binding pocket: Hydrophobic residues that accommodate aromatic catechol derivatives .

2.2. Sequence Homology

MhpB shares 58% sequence identity with the Alcaligenes eutrophus MpcI enzyme and forms a distinct class of extradiol dioxygenases. Conserved histidines in the N-terminal domain are critical for catalysis .

Catalytic Mechanism

MhpB employs a non-heme iron-dependent mechanism involving:

1. Substrate coordination: Catechol binds to Fe(II), enabling oxidative cleavage.

2. Lactone formation: A Criegee intermediate forms via radical recombination .

3. Ring fission: Hydrolysis of the lactone yields aliphatic dicarboxylic acids (e.g., 3-ketoadipic acid) .

Key Mutagenesis Findings:

• His-115 → Gln: Abolishes catalytic activity (loss of base).

• His-179 → Gln: Disrupts lactone hydrolysis (acidic group required).

• Asp-114 → Ala: Reduces activity at high pH (impacts metal chelation) .

Functional Roles in Catabolic Pathways

MhpB operates in convergent pathways for phenylpropanoid degradation:

1. PP pathway:

   • Hca enzymes oxidize PP to DHPP → MhpB cleaves DHPP → downstream mhp-encoded enzymes mineralize intermediates .

2. CI pathway:

   • Hca enzymes convert CI to DHCI → MhpB cleaves DHCI → mhp pathway further processes products .

Biotechnological Applications

MhpB’s ability to degrade aromatic pollutants (e.g., catechols, cinnamic acid derivatives) positions it for use in:

• Environmental remediation: Breaking down persistent aromatic contaminants .

• Industrial bioengineering: Modulating substrate preference via site-directed mutagenesis (e.g., enhancing DHCI specificity) .

Relevance to Photorhabdus luminescens subsp. laumondii

• Enhance biocontrol efficacy: By degrading plant-derived aromatic compounds in the rhizosphere .

• Expand metabolic versatility: Leveraging MhpB for aromatic compound catabolism in agricultural settings .

Research Gaps and Future Directions

• Recombinant expression: No studies document MhpB expression in P. luminescens.

• Synergistic systems: Combining MhpB with P. luminescens’ secondary metabolites (e.g., antibiotics) for dual-use applications.

• Structural optimization: Engineering MhpB for improved stability or substrate range using cryoEM-derived insights .