Enzymatic Synthesis of 2-Amino-adenosine Triphosphate as a Noncanonical Nucleotide Precursor of Z-Modified RNA

2-amino-deoxyadenosine (dZ) occurs naturally in certain bacteriophage genomes, where it replaces deoxyadenosine, forming three hydrogen bonds with thymidine. This noncanonical deoxyribonucleoside underlies the unique biophysical properties of dZ-DNA. Its corresponding ribonucleoside has been introduced to RNA to form Z-modified RNA, with promising applications in vaccine production and biomedicine. Unlike dZ-DNA, Z-modified RNA has only been synthesized in vitro, which required the addition of chemically synthesized 2-amino-adenosine triphosphate (ZTP) as a precursor. Here, we describe enzyme engineering studies on dZMP-succinate-synthetase (PurZ), a key enzyme in the bacteriophage dZ-DNA biosynthetic pathway that natively catalyzes the conversion of deoxyguanosine monophosphate (dGMP) to dZMP-succinate. Through site-saturation mutagenesis, we generated mutants with altered substrate specificity, capable of catalyzing the conversion of GMP to ZMP-succinate. We further demonstrated that these mutants, in combination with bacterial adenylosuccinate lyase, guanylate kinase and nucleoside diphosphate kinase, efficiently convert GMP to ZTP, marking a critical step in developing a biosynthetic pathway for Z-modified RNA, and enabling enzymatic synthesis of ZTP on a semipreparative scale. Our work provides the basis for further research on the impacts of Z-modified RNA in living organisms, and supports the cost-effective production of Z-modified RNA vaccines and therapeutics.