Biochemical characterization of a proline racemase from Loigolactobacillus bifermentans DSM 20003: identification of five N-terminal amino acid residues regulating oligomerization and catalytic activity.

Abstract

Proline racemase (ProR) catalyzes the reversible interconversion of L- and D-proline without a cofactor. We characterized two annotated ProR forms from Loigolactobacillus bifermentans DSM 20003 that differ in their predicted N-terminal starting positions. Lb-ProR2 exhibited high proline racemase activity and an apparent molecular mass consistent with a tetramer by size-exclusion chromatography (SEC), whereas Lb-ProR1, lacking five N-terminal residues, showed a major SEC peak consistent with a monomer. Sequential N-terminal deletions in Lb-ProR2 produced non-monotonic changes in activity and apparent oligomeric state. The first two deletion variants retained tetramer-like SEC behavior, whereas the third displayed monomer-like behavior and markedly reduced activity. Deletion of one additional residue restored tetramer-like behavior and partially recovered activity. AlphaFold and ESMFold modeling suggested preservation of the overall monomeric fold, although predicted N-terminal conformations varied with modeling context. Analysis of predicted tetramers identified N-terminal inter-subunit contacts and differences in contact patterns among variants. These results suggest that five N-terminal residues influence the apparent oligomeric organization and catalytic activity of Lb-ProR2, while the physiological translation initiation site remains to be established.

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