Published July 21, 2024
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Journal article
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Revealing reaction intermediates in one-carbon elongation by thiamine diphosphate/CoA-dependent enzyme family
Creators
- 1. University of Chicago
- 2. University of South Florida
- 3. Max Planck Institute for Terrestrial Microbiology
- 4. Argonne National Laboratory
- 5. Lawrence Berkeley National Laboratory
Description
2-Hydroxyacyl-CoA lyase/synthase (HACL/S) is a thiamine diphosphate (ThDP)-dependent versatile enzyme originally discovered in the mammalian α-oxidation pathway. HACL/S natively cleaves 2-hydroxyacyl-CoAs and, in its reverse direction, condenses formyl-CoA with aldehydes or ketones. The one-carbon elongation biochemistry based on HACL/S has enabled the use of molecules derived from greenhouse gases as biomanufacturing feedstocks. We investigated several HACL/S family members with high activity in the condensation of formyl-CoA and aldehydes, and distinct chain-length specificities and kinetic parameters. Our analysis revealed the structures of enzymes in complex with acyl-CoA substrates and products, several covalent intermediates, bound ThDP and ADP, as well as the C-terminal active site region. One of these observed states corresponds to the intermediary α–carbanion with hydroxymethyl-CoA covalently attached to ThDP. This research distinguishes HACL/S from related sub-families and identifies key residues involved in substrate binding and catalysis. These findings expand our knowledge of acyloin-condensation biochemistry and offer attractive prospects for biocatalysis using carbon elongation.
Data availability
The structural datasets generated during the current study are available in the Protein Data Bank repository (https://www.rcsb.org/) under accession codes: 8vzj, 8vzi, 8vzh, 8vze, 8vzd, 8vzk, 8vzf, 8vzc, 8vza, 8vzb. Plasmids for protein expression are available upon request. All other data generated during the current study including the raw kinetic and biophysical data are available upon request.Files
Revealing-reaction-intermediates-in-one-carbon-elongation.pdf
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Additional details
Identifiers
- DOI
- 10.1038/s42004-024-01242-y
- Other
- oai:uchicago.tind.io:12941
Funding
- Office of Science, U.S. Department of Energy
- DE-AC02-05CH11231
- U.S. Department of Energy
- DE-EE0008499