Abstract
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum. This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here, we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated, and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a post-translationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and Glu-236. The metalloprotein follows the halogenation cycle, whereby compound I oxidizes iodide and thiocyanate at high rates (>108 m−1 s−1) and bromide at very low rates. It is demonstrated that DdPoxA is up-regulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared with evolutionary-related mammalian peroxidases in the context of non-specific immune defense.
Highlights
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals
This work presents the first comprehensive biochemical study of an antibacterial peroxidase expressed in an organism that branched from the evolutionary tree close to the emergence of multicellularity
We focused on DdPoxA because D. discoideum is a social amoeba that can switch from a single-cell to a multicellular lifestyle under conditions of starvation
Summary
LPO, EPO, and MPO play an antimicrobial role by catalyzing the production of reactive oxidants, e.g. hypohalous acids or hypothiocyanate [10, 11]. LPO is secreted from mammary, salivary, and other mucosal glands; EPO is released by eosinophils, and MPO is secreted into the phagolysosome of phagocytosing neutrophils upon degranulation to kill engulfed pathogens such as bacteria [12,13,14]. The substantial similarity of the molecular mechanism(s) of phagocytosis and bacterial killing for food acquisition in D. discoideum and of the antimicrobial activity of neutrophils, monocytes, or macrophages [15, 16] prompted us to investigate the biochemistry and physiology of DdPoxA. We present the biochemical characterization and the first crystal structure of DdPoxA and demonstrate that the overall structures and heme cavity architecture of mammalian peroxidases were already established very early in evolution. It is demonstrated that the heme enzyme supports maintenance of sterility of the slug and the subsequently produced fruiting bodies, which suggests a role in antibacterial defense of the multicellular aggregate
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