Thiol-modifying inhibitors for understanding squalene cyclase function

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Squalene-hopene cyclase (SHC) is crucial for synthesizing hopanoids, essential membrane components in many bacteria.
• Understanding the precise mechanism of substrate access and catalysis in SHC is vital for elucidating bacterial lipid biosynthesis.

Purpose of the Study:

• To investigate the role of specific cysteine residues in the function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.
• To determine how substrate access to the active site is regulated and identify key residues involved in this process.

Main Methods:

• Site-directed mutagenesis was used to create mutants with altered cysteine residues.
• Thiol-reacting molecules and squalene analogues were employed as labeling agents and inhibitors.
• Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used to analyze modified enzyme fragments.

Main Results:

• Cysteine residue C435, located at a constriction in the substrate access channel, was identified as critical for enzyme function.
• Labeling experiments demonstrated that C435 is covalently modified by squalene-like inhibitors.
• Modification of C435 led to time-dependent inactivation of the enzyme, suggesting obstruction of the substrate channel.

Conclusions:

• Cysteine 435 plays a pivotal role in regulating substrate entry into the active site of squalene-hopene cyclase.
• The findings provide insights into the structural and functional importance of the nonpolar channel and its constriction.
• Targeting C435 with specific inhibitors offers a potential strategy for modulating SHC activity.