Unlocking a Nitrosuccinate Lyase for Decarboxylative Enzymatic Hydronitration.

Nitro compounds are central to synthetic chemistry, yet mild and selective biocatalytic routes to these motifs remain elusive. We report an enzymatic strategy for the unique decarboxylative hydronitration of fumarate, achieved by repurposing the nitrosuccinate lyase CreD from the aspartase/fumarase superfamily for the synthetic direction. CreD from Streptomyces cremeus and three related bacterial homologues catalyze hydronitration using sodium nitrite salt with remarkable efficiency (turnover numbers up to 102,000) and high atom economy. With an already exceptionally broad functional group tolerance across the superfamily, this feature underscores a conserved yet adaptable activity landscape. Guided by comprehensive mutagenesis and computational analysis across all functionally distinct superfamily members, we uncovered key molecular determinants that govern nucleophile selectivity and preserve the structural integrity required for active tetramer assembly. We also define a diagnostic fingerprint for predicting hydronitration activity and propose a reaction mechanism supported by extensive QM/MM simulations. These molecular insights provide a foundation for expanding biocatalytic Michael-type additions under environmentally benign aqueous conditions.