Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis

Significance

Itaconic acid was first described in the 19th century and was later appreciated mostly as a fungal metabolite of interest to polymer synthesis. Its surprising recent discovery as a key metabolite during the activation of inflammatory macrophages led to the recognition that it plays critical roles in linking metabolism and innate immunity. However, the lack of a crystal structure of cis-aconitate decarboxylase (CAD, the enzyme that synthesizes itaconate) has made it impossible to address many questions central to the chemistry, biology, evolution, and medical importance of itaconic acid synthesis. We have now determined the crystal structure of CAD and have identified amino acids that make up the active center, as well as human mutations with strong effects on itaconate synthesis.

Abstract

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus. We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus. Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus. We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.