Abstract
Bacterial poly(A) polymerases (PAPs) play an important role in RNA metabolism but remain poorly characterized outside Gammaproteobacteria. Here, we cloned and biochemically characterized the first PAP from Alphaproteobacteria, specifically from Marinobacter lipolyticus (Mli PAP). Using homology-based screening against E. coli PAP-1, we identified Mli PAP, sharing 54.8% sequence identity with its E. coli counterpart. The enzyme was expressed in E. coli but formed insoluble inclusion bodies; the active enzyme was purified as a fusion protein with the DsbA protein 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 K(m) comparable to E. coli PAP-1 (1.61 mM and 1.70 mM, respectively), and Mg(2+) (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 diversity and biochemical properties, as well as the potential applications of PAPs in biotechnology.