Molecules with nitrogen–nitrogen (N–N) bonds include diverse specialized metabolites from nature, but little is known about the underlying enzymatic mechanisms that have evolved for N–N bond formation. To directly form a single N(sp3)–N(sp3) bond, enzymes must reverse the typical nucleophilicity of one nitrogen. Here we report the structure of PipS, a haem-dependent enzyme that catalyses N–N bond formation in the cyclization of N5-OH-L-ornithine, giving L-piperazic acid. Our work reveals the role of a Lys–Thr dyad early in the mechanism and shows that PipS catalyses either N–N bond formation or imine-group formation in a substrate-specific manner, which may stem from a shared nitrenoid intermediate that effectively reverses the nucleophilicity of the hydroxylamine nitrogen. Our work expands knowledge of enzymatic N–N bond formation and delineates the catalytic versatility of a haem cofactor, paving the way for genetically encoded biocatalysts for N–N bond formation.
Dr. Garcia-Borràs is a co-corresponding author of the study, which was led by Prof. Katherine Ryan from University of British Columbia (Canada), recently reported in the Nature Catalysis journal. Dr. Jordi Soler (former PhD student in Garcia-Borràs’ group) is the first computational author of this publication.
This project is a core part of the research program that Dr. Garcia-Borràs leads at the IQCC, which is devoted to the use of computational methods in combination with experiments to characterize and design new abiological enzymatic activities and synthetically useful biocatalysts: “Biocatalytic intermediates for the discovery and design of new enzymatic activities“.
It has been recently published in Nature Catalysis:
Higgins, M.A., Shi, X. ‡, Soler, J. ‡, Harland, J.B., Parkkila, T., Lehnert, N., Garcia-Borràs, M.*, Du, Y.-L.*, Ryan, K.S*. (‡: these authors contributed equally)
“Structure and mechanism of haem-dependent nitrogen-nitrogen bond formation in piperazate synthase”
Nat. Catal., 2025, 8, 207–217.
DOI: 10.1038/s41929-024-01280-8
Girona, April 11th, 2025
For more info: ges.iqcc@udg.edu