Potential New Chemical Roles for Diheme Enzymes in Burkholderia

Abstract

Recent bioinformatics analysis led to the discovery of unreported diheme enzymes found in all strains of Burkholderia, which share sequence similarity to known bacterial cytochrome c peroxidases (bCCPs). bCCPs are periplasmic enzymes that reduce hydrogen peroxide (H2O2) to water. Traditional bCCPs contain a low potential, peroxidatic heme (LP) and a high‐potential heme (HP), which acts as a conduit for the delivery of reducing equivalents to the active site. Alternatively, within the bacterial diheme family there is MauG from Paracoccus denitrificans, an enzyme that utilizes peroxide to generate an unusual high valent bis‐Fe(IV) species required for the post translational modification of two tryptophan residues present in protein precursor, preMADH, to yield tryptophyltryptophan quinone (TTQ), the catalytic cofactor required for methylamine dehydrogenase (MADH). To explore how sequence divergence within the peroxidase superfamily reflects differences in functionality, we cloned, expressed and purified various diheme enzymes from Burkholderia. Of the two classes identified, BmvA, from B. multivorans and BpmB, from B. pseudomallei, show novel reactivity compared to bCCPs and MauG. BmvA has peroxidase activity comparable to bCCPs, yet nearly identical electron paramagnetic resonance (EPR) spectrum to MauG as well as a predicted bis‐Fe(IV) species, as identified by near‐infrared (NIR) spectroscopy. Unlike BmvA, BpmB has been observed to react in the presence of oxygen, suggesting novel O2 chemistry. Additionally, orthologs to these proteins in the non‐pathogenic strain B. thailandensis (BthA and BthB), behave similarly. We hypothesize that the sequence divergence of the Burkholderia enzymes correlates to the observed reactivity, ultimately introducing a new class of enzymes to the peroxidase superfamily.