Identification of a Novel Calcium Binding Motif Based on the Detection of Sequence Insertions in the Animal Peroxidase Domain of Bacterial Proteins

Abstract

Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33–79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca2+ binding with a KD of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

Extracellular haem peroxidases mediate Mn(II) oxidation in a marine Roseobacter bacterium via superoxide production

“…The AHPs of R . AzwK‐3b belong to a novel subfamily of bacterial peroxidases within the cyclooxygenase‐peroxidase superfamily (Zamocky et al ., ; Marchler‐Bauer et al ., ; Zámocký and Obinger, ; Santamaria‐Hernando et al ., ). In addition to the well‐known ‘animal’ peroxidases (e.g.…”

“…myeloperoxidases and thyroid peroxidases), the cyclooxygenase‐peroxidase superfamily contains peroxidases from all domains of life and was divided phylogenetically into seven subfamilies, at least three of which have prokaryotic representatives (Zámocký and Obinger, ). The specific subfamily that the AHPs in R. AzwK belong to have been identified in bacterial genomes of disparate phylogenies (Dick et al ., ; Zamocky et al ., ; Anderson et al ., 2009; 2011; Matilla et al ., ; Zámocký and Obinger, ; Santamaria‐Hernando et al ., ) (http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12893/suppinfo) and are distinguished from other identified peroxidases by repeat‐in‐toxin (RTX) family domains on the C‐terminal side of their putative haem‐binding sites (Zamocky et al ., ; Zámocký and Obinger, ; Santamaria‐Hernando et al ., ), as well as novel Ca 2+ ‐binding motifs within the catalytic domains (Santamaria‐Hernando et al ., ). The enzymes vary substantially in length and can have single or multiple haem‐binding sites and RTX‐family domains.…”

“…While it is becoming clear that these enzymes are able to generate Mn oxides, either directly or indirectly, this activity does not appear to be its primary role in the growth of this Roseobacter species. Indeed, while sequences for this AHP subfamily are widely distributed among taxonomically and ecologically diverse bacteria (http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12893/suppinfo) (Zamocky et al ., ; Santamaria‐Hernando et al ., ), Mn(II) oxidation has only been observed in a small subset of these organisms (e.g. http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12893/suppinfo), a further indication that Mn(II) oxidation is likely not their primary function in most organisms.…”

Heterologous Expression and Characterization of the Manganese-Oxidizing Protein from Erythrobacter sp. Strain SD21

“…Like MopA and the AHP domain, calcium binding is critical for activity of animal heme peroxidases (38). The residues that bind calcium in MPO and LPO are conserved in MopA and the AHP domain (39).…”

Comparative proteomics of Mn(II)‐oxidizing and non‐oxidizing Roseobacter clade bacteria reveal an operative manganese transport system but minimal Mn(II)‐induced expression of manganese oxidation and antioxidant enzymes

“…The lack of a clear differential expression of AHPs to Mn(II) would therefore suggest that Mn(II)-oxidizing activity is not upregulated by the presence of Mn(II) in this bacterium. Indeed, when examining the sequences of various bacterial AHPs, bacteria with known abilities to oxidize Mn(II) did not group together (Santamaria-Hernando et al, 2012). The variation in these sequences could therefore reflect a diverse range of enzymatic potential.…”