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.
- 1Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan. [email protected].
- 2Milk Science Research Institute, Megmilk Snow Brand Co., Ltd, Kawagoe, Japan.
Abstract
Background
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 13471T 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 Alrs and the VanT-type serine racemases.Results
Recombinant PdMurF'-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 Km values and reduced catalytic efficiency (kcat/Km), 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.Conclusions
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.
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BibTeX
@article{mutaguchi2026alanine,
title = {{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.}},
author = {Yuta Mutaguchi and Takuya Tsukahara and Shigeki Kada},
journal = {BMC microbiology},
year = {2026},
month = jul,
doi = {10.1186/s12866-026-05424-7},
url = {https://europepmc.org/article/MED/42972508},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - 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.
AU - Yuta Mutaguchi
AU - Takuya Tsukahara
AU - Shigeki Kada
PY - 2026
DA - 2026/07/27
JO - BMC microbiology
DO - 10.1186/s12866-026-05424-7
UR - https://europepmc.org/article/MED/42972508
AB - Background 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 13471T 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 Alrs and the VanT-type serine racemases.Results Recombinant PdMurF'-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 Km values and reduced catalytic efficiency (kcat/Km), 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.Conclusions 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.
SN - 1471-2180
ER -