Association Studies of Phage Metagenomes from Aquatic Environment Identify Known and Potentially New Key Players in the 2,6-Diaminopurine Biosynthesis Pathway

Advances in next-generation sequencing (NGS) and bioinformatics have expanded the potential of metagenomic studies, particularly for unculturable organisms such as phages with heavily modified genomes. In the evolutionary arms race between phages and their hosts, some phages develop a strategy to fully modify their DNAs to evade host-encoded antiphage defenses. Here, we developed a restriction-enzyme based method to selectively enrich all phages containing 2-aminoadenine (diaminopurine or dZ) directly from purified metagenomic DNA from environmental samples. Applying our previously established metagenomic genome-phenome association (metaGPA) pipeline, we identified 116 protein domains significantly associated with dZ phages. The top candidate domains fully recapitulated the phage-encoded dZ biosynthetic pathway. Study of individual enzymes, including PurZ and YfbR-like dATPase at residue resolution revealed key amino acids for the distinction between dZ and dA substrate. Additionally, we identified and validated two novel DNA polymerases with enhanced dZ incorporation activity and uncovered nucleoside 2-deoxyribosyltransferase (NDT) as a potential new component of the dZ biosynthetic pathway. Collectively, these findings refine current models of the dZ biosynthesis pathway, potentially reveal previously unrecognized enzymatic components, and expand the understanding of phage biology with implications for synthetic biology and DNA engineering.