Protein-Protein Complex Stability Controls Substrate Scope in a β-Ketoacyl-ACP Reductase Specific for Medium Chains

Area of Science:

• Biochemistry
• Molecular Biology
• Metabolic Engineering

Background:

• Assembly-line enzymes synthesize metabolites using acyl carrier proteins (ACPs) to shuttle intermediates.
• Controlling enzyme reaction order is crucial for metabolic engineering but remains poorly understood.

Purpose of the Study:

• Investigate the molecular basis of substrate specificity in β-ketoacyl-ACP reductase (PpFabG4).
• Explore how enzyme-ACP interactions influence substrate selection in fatty acid synthesis.

Main Methods:

• X-ray crystallography to analyze enzyme structure.
• Molecular simulations to study enzyme-ACP interactions.
• Site-directed mutagenesis to assess the impact of interaction strength on substrate specificity.

Main Results:

• PpFabG4 exhibits unusual medium-chain selectivity, not explained by active site accessibility.
• Substrate preference is dictated by the stability of the enzyme-ACP interaction, which is enhanced by medium acyl chains.
• Mutational studies demonstrated over 100-fold changes in short-chain substrate activity by altering enzyme-ACP interaction strength.

Conclusions:

• Enzyme-ACP interaction stability is a key determinant of substrate scope in promiscuous enzymes.
• Findings provide a mechanism for controlling intermediate exchange in assembly-line systems.
• This work guides the reprogramming of enzymatic pathways for biosynthetic chemistry and metabolic engineering.