Redirecting P450 EryK specificity by rational site-directed mutagenesis

Area of Science:

• Biochemistry
• Enzyme Engineering
• Antibiotic Biosynthesis

Background:

• EryK, a cytochrome P450, is crucial for erythromycin A (ErA) biosynthesis.
• EryK's strict substrate specificity leads to the accumulation of the shunt metabolite ErB, reducing ErA yield and purity.
• ErB is a byproduct with limited antimicrobial activity and moderate toxicity.

Purpose of the Study:

• To redesign EryK for industrial applications by enabling it to process the ErB metabolite.
• To investigate the structural and catalytic effects of active-site mutations on EryK's substrate specificity.

Main Methods:

• Utilized the three-dimensional structure of EryK complexed with ErD to design active-site mutants.
• Created three single active-site mutants: M86A, H88E, and E89L.
• Analyzed the binding and catalytic properties of these mutants on both ErD and ErB.

Main Results:

• The M86A mutation in EryK demonstrated enzymatic activity on both ErB and ErD.
• Structural analysis revealed the M86A mutation allows ErB to fit and induce closure of the enzyme's active site.
• This active site closure facilitates the catalytic reaction, recovering fine substrate recognition and locking.

Conclusions:

• A single point mutation (M86A) successfully engineered EryK to hydroxylate the non-physiological substrate ErB.
• This engineered EryK variant enhances ErA production by processing the byproduct ErB.
• The study demonstrates the potential of rational enzyme design for improving industrial antibiotic manufacturing.