A Split Gene Approach to Alleviate Severe Inhibition of Catalysis by Substrate

Area of Science:

• Biocatalysis and Enzyme Engineering
• Environmental Biotechnology
• Protein Structure-Function Relationships

Background:

• Iodotyrosine deiodinases (IYDs) are crucial enzymes involved in thyroid hormone metabolism.
• Substrate inhibition limits the catalytic efficiency of many IYDs, including Thermotoga neapolitana IYD (TnIYD).
• TnIYD is a target for engineering robust dehalogenation of persistent environmental pollutants like halophenols.

Purpose of the Study:

• To elucidate the mechanism of substrate inhibition in TnIYD and its human homologue.
• To engineer a novel TnIYD variant that overcomes substrate inhibition for enhanced halophenol degradation.
• To investigate the role of the active site lid in TnIYD catalysis.

Main Methods:

• Determined the mechanism of substrate inhibition via formation of a nonproductive complex between 3-iodo-l-tyrosine and oxidized TnIYD.
• Employed a split gene coexpression approach to generate TnIYD fragments that assemble into an active enzyme.
• Engineered three sets of enzymes with discontinuities and deletions in the active site lid.

Main Results:

• Identified substrate inhibition in TnIYD and human IYD arises from a nonproductive complex preventing enzyme reduction.
• At least one engineered TnIYD variant successfully overcame substrate inhibition.
• The engineered enzyme demonstrated a significant increase in catalytic rate (kcat), indicating enhanced dehalogenation efficiency.

Conclusions:

• A split gene approach effectively overcomes substrate inhibition in TnIYD.
• A substantial portion of the active site lid is dispensable for reductive dehalogenation.
• This strategy provides a novel method for enzyme engineering, complementing circular permutation techniques.