2024 / The di-heme bacterial cytochrome c peroxidase (BCcP) superfamily can be divided into two groups based on biological functions. Canonical BCcPs reduce hydrogen peroxide to water by taking electrons from their redox partner - cytochrome c. In contrast, MauG and its orthologs are involved in protein post-translational modifications using the bis-Fe (IV) species. One of the key factors that is highly corelated with the diversity of biological functions of BCcPs is the distal heme ligand on the electron transfer heme (E heme), demonstrating the significant structure-function relationships of BCcPs. Using the sequence similarity network coupled with genome neighborhood analysis, a new group of BCcPs has been discovered from marine bacteria. The sequence analysis indicates the lack of distal ligand on E heme. In addition, the conserved genome context consisted of PhnDCE phosphonate transporter and manganese dependent transcriptional repressor MntR suggests potential functions in phosphorus metabolism under metal dependent regulation, which is beyond the current understanding of the functions of BCcP enzymes. The biochemical and biophysical characterization of RolA, the newly discovered BCcP from Roseovarius lutimaris, reveals that it possesses no peroxidase activity but reacts with O2 when fully reduced, setting it apart from all known BCcPs. In order to investigate the biological function of RolA, genetic manipulation tools have been developed in R. lutimaris. Three deletion mutants including R. lutimaris ∆rolA, ∆mntR and ∆rolA-∆mntR have been generated. The qPCR analysis of R. lutimaris WT vs ∆mntR has shown up-regulation of rolA upon mntR deletion, confirming the transcription repression of RolA by MntR. Native RolA has been tagged and over-expressed in R. lutimaris. The UV-vis and EPR spectra have demonstrated that the heme environment of native RolA is different from known BCcPs. Like recombinant RolA, native RolA has no peroxidase activity and reacts with O2 under fully reduced state. Preliminary studies using R. lutimaris ∆rolA and ∆rolA-∆mntR have shown utilization of phosphonates as sole phosphorus source. Bioinformatics analysis of PhnD from R. lutimaris suggests RolA might be related with hypophosphite transformation. The investigation of biological function of RolA using growth assay and in vitro enzymatic activity assay is ongoing. The identification of RolA expands the knowledge and diversity of the BCcP superfamily. / 2027-02-12T00:00:00Z