Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Site-specific covalent conjugation is crucial for studying protein-protein interactions.
• Understanding enzyme-substrate interactions is vital for metabolic pathway elucidation.

Purpose of the Study:

• To investigate the interaction between Escherichia coli acyl carrier protein (AcpP) and BioF using sulfur fluoride exchange (SuFEx) chemistry.
• To identify the specific crosslinking site and analyze the structural and functional basis of the BioF-AcpP complex.

Main Methods:

• Site-specific covalent crosslinking using SuFEx warheads.
• Mass spectrometry/mass spectrometry (MS/MS) for crosslink site identification.
• Protein crystallography and site-directed mutagenesis for interface analysis.

Main Results:

• SuFEx successfully crosslinked AcpP with BioF at a tyrosine residue near the active site.
• MS/MS analysis pinpointed the crosslinking site within the peptide originating from both proteins.
• Crystallography and mutational studies identified three critical arginine residues on BioF involved in AcpP recognition.
• These interactions facilitate pimeloyl-AcpP acting as an acyl donor for PLP-mediated catalysis.

Conclusions:

• The study demonstrates SuFEx as an effective tool for probing protein-protein interactions in biotin biosynthesis.
• Identified BioF-AcpP interface residues and interactions provide mechanistic insights into biotin synthesis.
• Findings support an evolutionary gain-of-function for BioF, enabling direct biotin synthesis from fatty acid precursors.