Crossing Enzymatic Boundaries by Coupling BchNB with the Nitrogenase Cofactor Precursor

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Photosynthesis and Nitrogen Fixation

Background:

• The dark-operative protochlorophyllide oxidoreductase (DPOR) is crucial for chlorophyll biosynthesis, catalyzing the light-independent reduction of protochlorophyllide to chlorophyllide.
• DPOR shares structural and mechanistic similarities with nitrogenase, the enzyme responsible for nitrogen fixation, suggesting a potential evolutionary relationship.

Purpose of the Study:

• To investigate the structural homology between DPOR and nitrogenase components.
• To engineer a chimeric metalloenzyme by combining DPOR and nitrogenase components.
• To explore the catalytic potential of the engineered enzyme for nitrogen and carbon substrate reduction.

Main Methods:

• Structural alignment of Rhodobacter capsulatus BchNB (DPOR catalytic component) with Azotobacter vinelandii NifDK (nitrogenase catalytic component) and NifEN (cofactor maturase).
• Reconstitution of Pchlide-free RcBchNB with the L-cluster from AvNifEN to create a hybrid enzyme (RcBchNB^L).
• Enzymatic assays using N₂, CN^-, and CO as substrates in the presence of a strong reductant (Eu^II-DTPA).

Main Results:

• A conserved α₂β₂ architecture and a shared cofactor-insertion pathway were identified between DPOR and nitrogenase components.
• The engineered hybrid enzyme RcBchNB^L demonstrated catalytic activity in reducing N₂ to NH₃ and C₁ substrates (CN^-, CO) to hydrocarbons.
• Competition between Pchlide and the L-cluster for a common binding site was observed, impacting hybrid enzyme activity.

Conclusions:

• The findings support an intertwined evolutionary history of photosynthetic and nitrogen-fixing enzymes.
• A framework for engineering chimeric metalloenzymes with coupled light-harvesting and nitrogenase-like catalytic functions is proposed.
• This research opens avenues for creating novel biocatalysts with tailored reactivities.