Functional Identification of Rubber Oxygenase (RoxA) in Soil and Marine Myxobacteria

Abstract

ABSTRACTThe rubber oxygenase (RoxA) ofXanthomonassp. strain 35Y (RoxAXsp) is so far the only known extracellularc-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 ofHaliangium ochraceum,Myxococcus fulvus,Corallococcus coralloides, andChondromyces apiculatus. TheroxAorthologs ofH. ochraceum(RoxAHoc),C. coralloidesBO35 (RoxACco), andM. fulvus(RoxAMfu) were functionally expressed in a ΔroxA Xanthomonassp. 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 RoxACcowere purified and biochemically characterized. The optimal temperature of RoxACcoand RoxAMfuwas between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxACcoand RoxAMfuindicated 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 RoxAXspstructure 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 theXanthomonassp. 35Y,H. ochraceum,M. fulvus, andC. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other thanXanthomonasproves that RoxA is more common among rubber degraders than was previously assumed.