Humans have been using enzymes to create new products for thousands of years. First it was wine, then cheese. In this tradition, three years ago, a team of scientists tweaked a lyase (HACL/S) to reverse course. Instead of breaking, the enzyme synthesizes novel chemicals through the addition of carbon atoms.
Now, using the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory, an international team shows how HACL/S enzymes work on an atomic level. Their findings can serve as the basis for increasing the enzymes’ yield and versatility while drawing down as precursors atmospheric carbon dioxide and methane.
HACL/S enzymes were originally discovered for their role in breaking down fatty acids into formyl-CoA (fCoA) and an aldehyde or ketone in mammalian peroxisomes. Since then, scientists have discovered their ability to condense fCoA with various aldehydes and ketones and have one carbon atom added to them. Given the enzyme’s ability to reverse reaction direction from a lyase to a synthase, combined with an abundance of carbon molecules in the atmosphere, HACL/S is an ideal model for biocatalytic production of a variety of new products.
However, compared to chemical synthetic reactions, biocatalytic production usually produces low yield. The authors of the current research reasoned that if they could manipulate the specificity of these enzymes to accept different kinds of ketones or aldehydes, they could boost the enzymes’ productivity and efficiency.
In order to do that, they first needed to discover how these enzymes worked.
To begin, the team chose from the list of over 100 newly identified proteins six variants of the enzyme that exhibited high activity with aldehyde compounds of different length and formyl-CoA and had amino acid sequences that were diverse enough to cover the HACL/S subfamily. The team synthesized genes for each of the variants, then expressed them in Escherichia coli bacteria.
After purifying the expressed proteins, some members of the international team characterized the enzymes biochemically. Others produced crystals of five enzymes separately and in complexes with acyl-CoA substrates, ThDP cofactor, and ADP. They X-rayed the crystals, diffracted to 1.70–2.70 Å, at beamlines 19-ID – the Structural Biology Center (SBC) – and 23-ID-B – the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) – of the Advanced Photon Source (APS) at Argonne National Laboratory.
The crystal structures obtained from the X-ray data revealed what computer-predicted models could not: a flexible loop on the C terminus that locked on the cofactor and kept it bound to the enzyme’s active site. When the substrate was added, the loop closed the active site, stabilizing the cofactor and enabling the transfer of the formate compound to the substrate.
Read more on Argonne website
