The First Poly(A) Polymerase from Alphaproteobacteria

Bacterial poly(A) polymerases (PAPs) play important role in RNA metabolism but re-main poorly characterized outside Gammaproteobacteria. Here, we cloned and biochemi-cally characterized the first PAP from Alphaproteobacteria, specifically from Marinobacter lipolyticus (Mli PAP). Using homology-based screening against E. coli PAP-1, we identi-fied Mli PAP, sharing 54.8% sequence identity with its E. coli counterpart. The enzyme was expressed in E. coli but formed insoluble inclusion bodies; active enzyme was solu-bilized and used for functional assays. Mli PAP exhibited optimal activity at 30°C and similar thermostability to E. coli PAP-1. ATP was the preferred substrate, with Km com-parable to E. coli PAP-1 (1.61 mM and 1.70 mM, respectively), and Mg²⁺ (10 mM) was identified as the optimal cofactor. Mli PAP displayed salt-dependent activity, with the most effective polyadenylation in KCl and inhibition by NaCl and ammonium salts, contrasting with the halophilic nature of its host. This study provides the first functional insights into PAPs from Alphaproteobacteria, broadening the understanding of PAP diver-sity and biochemical properties, potential applications of PAPs in biotechnology.