Structural and Functional Characterization of Mycobacterium tuberculosis Homoserine Transacetylase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Mycobacterium tuberculosis (Mtb) homoserine transacetylase (HTA) is crucial for methionine biosynthesis and protein synthesis.
• Mtb HTA deficiency leads to toxic lysine accumulation and impaired S-adenosyl-l-methionine-dependent enzyme activity.
• Previous studies validated Mtb HTA as an essential enzyme with a ligandable binding site.

Purpose of the Study:

• To elucidate the mechanistic details and structural characteristics of Mtb HTA.
• To provide a foundation for structure-based drug design against Mtb.

Main Methods:

• Crystallography and mass spectrometry to observe the acetylated HTA intermediate.
• Initial velocity and kinetic characterization of wild-type HTA and site-directed mutants with various substrates.
• Fluorescence quenching titrations to analyze substrate binding interactions.

Main Results:

• A ping-pong kinetic mechanism was identified for Mtb HTA.
• Substrate specificity and critical catalytic residues were determined through kinetic analysis.
• Acetyl-CoA and L-homoserine exhibit distinct binding affinities, and feedback inhibition mechanisms involving acetyl-CoA, CoA, and O-acetyl-L-homoserine were proposed.

Conclusions:

• Detailed mechanistic and structural insights into Mtb HTA were obtained.
• These findings support Mtb HTA as a promising target for structure-based drug development against tuberculosis.
• Understanding HTA's regulation provides avenues for therapeutic intervention.