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Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS
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Structural basis for substrate specificity and MSMEG_0435-0436 binding by the mycobacterial long-chain acyl-CoA
Yingke Liang1,2, Stephanie A Bueler1, John L Rubinstein1,2,3
1Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
Summary
The long-chain acyl-coenzyme A (CoA) carboxylase (LCC) complex in mycobacteria, essential for cell wall biosynthesis, has been structurally characterized. New insights reveal how its subunits achieve substrate specificity and how regulatory proteins modulate activity.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Mycolic acids are key components of the mycobacterial cell wall, forming a barrier against antibiotics.
- The long-chain acyl-coenzyme A (CoA) carboxylase (LCC) complex is essential for synthesizing precursors for mycolic acid and tuberculostearic acid.
- The LCC complex comprises subunits AccA3 (with biotin carboxylase and biotin carboxyl carrier protein domains), AccD4, AccD5, and AccE.
Purpose of the Study:
- To determine the high-resolution structures of the LCC complex from *Mycobacterium smegmatis*.
- To elucidate the mechanisms of substrate specificity and carboxyl transfer within the LCC complex.
- To investigate the function of regulatory proteins that interact with the LCC complex.
Main Methods:
- Electron cryomicroscopy (cryo-EM) was used to determine the structures of the LCC complex.
- Cryo-EM was employed to capture the enzyme during catalysis to observe substrate binding and transfer.
- Biochemical assays were performed to assess the activity of the LCC complex in the presence of regulatory proteins.
Main Results:
- The LCC complex structure reveals a core of AccD4 and AccD5 flanked by AccA3 subunits, tethered by AccE.
- AccD5 exhibits high affinity for CoA, while AccD4 binds long acyl chains, defining substrate specificity.
- Biotin carboxyl carrier protein domains translocate over long distances to transfer carboxyl groups.
- A regulatory complex (MSMEG_0435/MSMEG_0436) binds the LCC, sequestering biotin moieties and reducing propionyl-CoA carboxylase activity.
Conclusions:
- The structural and mechanistic insights into the LCC complex provide a foundation for understanding mycobacterial cell wall biosynthesis.
- The identified regulatory proteins, including the *M. tuberculosis* ortholog Rv0263c, may play a role in controlling fatty acid precursor production.
- Understanding these pathways could offer new targets for antibiotic development against *Mycobacterium* species.
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