An alanine racemase with a novel domain architecture: a chimeric enzyme with an N-terminal Mur ligase-like domain in Phocaeicola dorei and its distribution in the phyla Bacteroidota and Chloroflexota

Alanine racemase (Alr) catalyzes the interconversion of l -alanine and d -alanine, supplying the d -alanine required for bacterial peptidoglycan synthesis. We identified a previously unreported alr gene in Phocaeicola dorei JCM 13471 T encoding a chimeric protein consisting of an N-terminal MurF ligase-like domain (MurF’) fused to a C-terminal Alr domain. This domain architecture represents a third structural type of bacterial Alr, distinct from canonical Alr and the VanT-type serine racemase. Recombinant Pd MurF’-Alr preferentially catalyzed the racemization of l - and d -alanine and exhibited only weak activity toward serine. N-terminal truncation analyses demonstrated that the MurF’ region substantially influenced the catalytic properties of the Alr domain. Complete deletion of MurF’ markedly increased the K m values and reduced catalytic efficiency ( k cat / K m ), indicating that MurF’ enhances both substrate affinity and catalytic activity. Database searches showed that putative MurF’-Alr homologues or related sequences are predominantly distributed in the bacterial phyla Bacteroidota and Chloroflexota . Phylogenetic analysis revealed contrasting evolutionary patterns between these phyla. In Chloroflexota , MurF’ sequences clustered with coexisting non-chimeric MurF proteins, whereas in Bacteroidota they formed a distinct clade. By contrast, the Alr domains of MurF’-Alr proteins in Bacteroidota were interspersed among non-chimeric Alr proteins rather than forming a single clade, consistent with repeated fusion of murF -like sequences with pre-existing alr genes or recurrent gain and loss of the MurF’ region. This study identifies and biochemically characterizes a previously unknown MurF’-Alr chimeric enzyme from P. dorei . The MurF’ region may function as a modulator of alanine racemase activity rather than simply serving as an additional structural domain. Our phylogenetic analyses suggest that MurF’-Alr has undergone distinct evolutionary trajectories in Bacteroidota and Chloroflexota . This study provides new insights into the evolution and functional diversification of bacterial enzymes involved in d -alanine biosynthesis.