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

摘要

The rubber oxygenase (RoxA) of Xanthomonas sp. strain 35Y (RoxA_( Xsp )) 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 (RoxA_( Hoc )), C. coralloides BO35 (RoxA_( Cco )), and M. fulvus (RoxA_( Mfu )) were functionally expressed in a Δ roxA 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. RoxA_( Xsp ), RoxA_( Mfu ), and RoxA_( Cco ) were purified and biochemically characterized. The optimal temperature of RoxA_( Cco ) and RoxA_( Mfu ) was between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxA_( Cco ) and RoxA_( Mfu ) indicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxA_( Xsp ). Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxA_( Xsp ) structure and included several conserved residues, notably, W_(302), F_(317), and a MauG motif at about H_(517). 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.