ABSTRACT
Non-ribosomal peptide synthetases (NRPSs) and NRPS-like enzymes are abundant in microbes as they are involved in the production of primary and secondary metabolites. In contrast to the well-studied NRPSs, known to produce non-ribosomal peptides, NRPS-like enzymes exhibit more diverse activities and their evolutionary relationships are unclear. Here, we present the first in-depth phylogenetic analysis of fungal NRPS-like A domains from functionally characterized pathways, and their relationships to characterized A domains found in fungal NRPSs. This study clearly differentiated amino acid reductases, including NRPSs, from CoA/AMP ligases, which could be divided into 10 distinct phylogenetic clades that reflect their conserved domain organization, substrate specificity and enzymatic activity. In particular, evolutionary relationships of adenylate forming reductases could be refined and explained the substrate specificity difference. Consistent with their phylogeny, the deduced amino acid code of A domains differentiated amino acid reductases from other enzymes. However, a diagnostic code was found for α-keto acid reductases and clade 7 CoA/AMP ligases only. Comparative genomics of loci containing these enzymes revealed that they can be independently recruited as tailoring genes in diverse secondary metabolite pathways. Based on these results, we propose a refined and clear phylogeny-based classification of A domain-containing enzymes, which will provide a robust framework for future functional analyses and engineering of these enzymes to produce new bioactive molecules.