Abstract
Amino acid racemases catalyze the interconversion of L- and D-amino acids, maintaining intracellular levels of both D- and L-amino acids. While alanine and glutamate racemases are widespread in bacteria, serine racemase (SerR) is predominantly found in animals. Recently, homologs of animal SerR were reported in some bacterial genomes, but their evolutionary distribution and functional roles remain poorly understood. In this study, we cloned and expressed 20 SerR homologous genes from 13 bacterial species spanning five phyla and characterized their enzymatic activity. Six homologs exhibited serine dehydratase activity, while the remaining showed racemase activity with serine, aspartate, asparagine, or arginine. Notably, the SerR homologs from Parafannyhessea umbonata (Actinomycetota), Clostridium aceticum, Anaerovirgula multivorans, Alkaliphilus oremlandii (Bacillota), Acetomicrobium mobile, and Thermovirga lienii (Synergistota) demonstrated strong arginine racemase activity, with K(m) values ranging from 0.167 to 0.885 mM and k(cat) values ranging from 5.86 to 61.5 s(-1) for L-arginine. Phylogenetic analysis revealed that bacterial and eukaryotic SerR homologs share a common ancestral gene, and substrate specificity has independently changed multiple times during evolution. Amino acid sequence alignment and analysis of site-directed mutants revealed that residues at positions 146 to 148 and surrounding regions, located near the substrate-binding site, play a crucial role in substrate specificity and/or catalytic activity. These results highlight the evolutionary processes that drive functional diversification in serine racemase homologs.