The rubber oxygenase (RoxA) of Xanthomonas sp. strain 35Y (RoxAXsp) is so far the only known extracellular c-type diheme cytochrome that is able to cleave poly(cis-1,4-isoprene). All other rubber-degrading bacteria described are Gram positive and employ a nonheme protein (latex-clearing protein [Lcp]) for the postulated primary attack of polyisoprene. Here, we identified RoxA orthologs in the genomes of Haliangium ochraceum, Myxococcus fulvus, Corallococcus coralloides, and
Chondromyces apiculatus. The roxA orthologs of H. ochraceum (RoxAHoc), C. coralloides BO35 (RoxACco), and M. fulvus (RoxAMfu) were functionally expressed in a ΔroxA Xanthomonas sp. 35Y background. All RoxA orthologs oxidatively cleaved polyisoprene, as revealed by restoration of clearing-zone formation and detection of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as a cleavage product. RoxAXsp, RoxAMfu, and RoxACco were purified and biochemically characterized. The optimal temperature of RoxACco and RoxAMfu was between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxACco and RoxAMfu indicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxAXsp. Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxAXsp structure and included several conserved residues, notably, W302, F317, and a MauG motif at about H517. Lcp-like sequences were not detected in the genomes of the Xanthomonas sp. 35Y, H. ochraceum, M. fulvus, and C. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other than Xanthomonas proves that RoxA is more common among rubber degraders than was previously assumed.