Binding and Translocation of Substrate Allosterically Promotes Functional Interactions Within the AlkB-AlkG Electron...

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• The alkane monooxygenase (AlkB) and rubredoxin (AlkG) complex catalyzes alkane hydroxylation.
• A recent cryo-EM study detailed the Fontimonas thermophila AlkB-AlkG complex structure with a dodecane (D12) substrate.
• The precise molecular mechanism of this complex remains largely unelucidated.

Purpose of the Study:

• To investigate the dynamics and interactions within the Fontimonas thermophila AlkB-AlkG complex.
• To elucidate the molecular mechanism of alkane hydroxylation and substrate translocation.
• To identify key sites and interactions for potential engineering of AlkB variants.

Main Methods:

• Multiscale computations, including molecular dynamics (MD) simulations.
• Elastic network models (ENM) for allosteric effect analysis.
• Quantum mechanics/molecular mechanics (QM/MM) for oxygen activation mechanism.

Main Results:

• Dodecane (D12) substrate exhibited stable interactions in the active site during MD simulations.
• A hydrophobic channel, gated by I54, was identified, allowing D12 translocation to a membrane-exposed site via intermediate sites IS1 and IS2.
• Substrate translocation influences the coupling between iron centers and the AlkB-AlkG interface, with channel connectivity to O2 passage sites.
• ENM analysis confirmed allosteric effects between substrate entry, catalytic site, and the AlkB-AlkG interface.

Conclusions:

• The study reveals a dynamic mechanism for substrate translocation in the AlkB-AlkG complex.
• Identified key residues and pathways involved in substrate binding, translocation, and allosteric regulation.
• Provides mechanistic insights for engineering AlkB variants with enhanced alkane conversion capabilities.