Structural insights into interdomain interactions in Entamoeba histolytica APS kinase

• The structure of adenosine 5′-phosphosulfate kinase, a key enzyme in the uniquely evolved sulfate activation pathway of Entamoeba histolytica , was newly determined. • Additional x-ray crystallography of a mutant and single-particle cryo-EM of full-length identified interdomain interactions not previously reported in other organisms. • Activity assays of mutants suggest the presence of a regulatory mechanism mediated by these interdomain interactions. The biosynthetic pathway of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is a universal and essential metabolic process in many organisms, providing the activated sulfate donor required for the synthesis of diverse sulfated metabolites. However, this pathway has undergone substantial evolutionary diversification among species. In Entamoeba histolytica , PAPS biosynthesis occurs within the mitosomes, mitochondrion-related organelles (MROs), representing a distinctive example of lineage-specific evolutionary adaptation. PAPS synthesis proceeds through a conserved two-step process, which is sequentially catalyzed by ATP sulfurylase (AS) and adenosine 5′–phosphosulfate (APS) kinase (APSK). In this study, we focused on E. histolytica APSK ( Eh APSK). Eh APSK contains an additional AS-like domain (SLD), although its functional role remains unclear. Here, we determined the crystal structure of full-length Eh APSK at 2.60 Å resolution and the structure of the truncated Eh APSK lacking APS kinase domain (KD) ( Eh APSK ΔKD ) at 2.10 Å resolution. Structural analyses revealed that the SLD engages in dynamic contacts with the KD. Furthermore, deletion of the domain and mutational analyses indicated that the SLD significantly influences the catalytic activity of the KD. Based on these findings, we propose a new regulatory mechanism in which transient interdomain interactions modulate APS kinase activity, representing an unique evolutionary adaptation of E. histolytica .