Revealing reaction intermediates in one-carbon elongation by thiamine diphosphate/CoA-dependent enzyme family

Youngchang Kim; Seung Hwan Lee; Priyanka Gade; Maren Nattermann; Natalia Maltseva; Michael Endres; Jing Chen; Philipp Wichmann; Yang Hu; Daniel G Marchal; Yasuo Yoshikuni; Tobias J Erb; Ramon Gonzalez; Karolina Michalska; Andrzej Joachimiak

doi:10.1038/s42004-024-01242-y

Revealing reaction intermediates in one-carbon elongation by thiamine diphosphate/CoA-dependent enzyme family

Commun Chem. 2024 Jul 21;7(1):160. doi: 10.1038/s42004-024-01242-y.

Authors

Youngchang Kim^{1

2}, Seung Hwan Lee³, Priyanka Gade², Maren Nattermann⁴, Natalia Maltseva², Michael Endres¹, Jing Chen³, Philipp Wichmann⁴, Yang Hu³, Daniel G Marchal⁴, Yasuo Yoshikuni⁵, Tobias J Erb^{4

6}, Ramon Gonzalez⁷, Karolina Michalska^{8

9}, Andrzej Joachimiak^{10

11

12}

Affiliations

¹ eBERlight and Structural Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, IL, USA.
² Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA.
³ Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
⁴ Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
⁵ The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. yyoshikuni@lbl.gov.
⁶ Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.
⁷ Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA. ramongonzale@usf.edu.
⁸ eBERlight and Structural Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, IL, USA. kmichalska@anl.gov.
⁹ Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA. kmichalska@anl.gov.
¹⁰ eBERlight and Structural Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, IL, USA. andrzejj@anl.gov.
¹¹ Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA. andrzejj@anl.gov.
¹² Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. andrzejj@anl.gov.

Abstract

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.

Abstract

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