Introduction:

The acetyl-CoA decarbonylase/synthase (ACDS) enzyme complex plays a key role in many acetogenic bacteria and methanogenic archaea. As part of the ancient Wood-Ljungdahl pathway, it catalyses the reversible formation of acetyl-CoA from CO2, a methyl group, coenzyme A and reduced ferredoxin. In acetoclastic methanogens, this reaction runs in reverse leading to the formation of a methyl group which is further transferred and reduced to build up a Na+/H+ gradient for ATP-synthesis. Distinct from its bacterial homolog, the archaeal ACDS forms a ~2 MDa superassembly of so-far unknown structure.

Goals:

We want to determine the three-dimensional structure of the archaeal ACDS complex and resolve catalytically important conformations within the assembly. In the end, we would like to understand the functional differences between the bacterial and archaeal complex.

Materials & Methods:

We are working with the ACDS complex natively and anaerobically purified from Methanosarcina acetivorans. Since previous approaches to determine the structure of ACDS by cryo-EM single-particle analysis yielded poor resolutions, we employ an integrative structural biology approach with cross-linking mass spectrometry and mass photometry to characterise subcomplexes of the superassembly and their interactions.

Results:

After successfully disassembling the ACDS complex into three smaller, functional subcomplexes, we found that they form different homo-oligomers. Furthermore, we identified terminal disordered regions of three protein subunits as likely being responsible for the oligomerisation patterns. Using cross-linking mass spectrometry, we resolved two catalytically relevant conformations of the acetyl-CoA synthase subcomplex bound to either one of the other two subcomplexes.

Summary:

The archaeal ACDS complex displays a molecular architecture which is clearly distinct from its bacterial counterpart. However, the exact functional difference as well as the structure of this complex are largely not understood despite three decades of research. With our work, we hope to gain further insights into this puzzle.

Further reading: https://doi.org/10.1021/acs.biochem.2c00425