DISCUSSION

Biosynthetic natural rubber and chemosynthetic rubber have been used by humans at a level of about 10⁶ tons/year for about a century. The intrinsic biodegradability of polyisoprene by microorganisms in the environment has been known since the first publication of Söhngen and Fol in 1914 . Many other reports on biodegradation of polyisoprene have appeared during the last few decades. In particular, the number of isolated rubber-degrading microorganisms has increased during the last 10 years . The biochemical mechanism by which the rubber backbone is cleaved is only poorly understood, however, and until now not one enzyme involved in rubber degradation has been isolated.

In this study we purified an extracellular protein from polyisoprene-grown Xanthomonas sp. cultures (RoxA) that has the ability to cleave the carbon backbone of polyisoprene in vitro, and we characterized its biochemical properties. A low-molecular-mass compound derived from a three-isoprene-unit backbone (12-oxo-4,8-dimethyltrideca-4,8-diene-1-al; m/z 236) was identified as the major degradation end product of in vitro rubber degradation by purified RoxA. Four additional minor products that differed by a mass increment of m/z n x 68 from the main metabolite (m/z 236) were characterized in the corresponding ion chromatograms by HPLC-negative ESI-MS analysis. Since the repetitive unit in polyisoprene has a molecular mass of 68 Da, it can be safely assumed that the minor products have the same functional groups as the main metabolite (m/z 236) and that the only structural difference is the number of isoprene units, CH₂-C(CH₃)CH-CH₂, incorporated between the terminal functional groups. The concentration of these oligomers was 1 to 2 orders of magnitude lower than that of ODTD. ODTD thus apparently is the principal end product of the RoxA-catalyzed cleavage of polyisoprene. These findings are consistent with the assumption that RoxA cleaves polyisoprene oxidatively at regular intervals, cutting off three isoprene units per step. Our results are in good agreement with previous findings of Tsuchii and coworkers, who identified a whole range of related oligomers with more than 100 isoprene units in addition to ODTD . However, these results were obtained with undefined culture fluid, and it was not known how many enzymes were involved. Presumably, the concentration and/or activity of RoxA in the culture fluid in the experiments of Tsuchii et al. was not high enough to allow complete degradation of polyisoprene.

Trypsin fingerprint analysis of RoxA confirmed that RoxA is identical to the product of a recently cloned gene assumed to be involved in rubber degradation . The presence of a functional signal sequence in the cloned gene was in agreement with the extracellular localization of RoxA. Comparison of the amino acid sequence of RoxA deduced from the gene with the database revealed the presence of several related amino acid sequences of hypothetical proteins. In addition to related sequences found previously , sequences coding for a hypothetical protein of Pirellula sp. (gi32473529), hypothetical protein Bd3821 of Bdellovibrio bacteriovorus (gi42525145), and some hypothetical proteins deduced from sequences of environmental samples were found. A function has not been identified for any of the related proteins; however, the RoxA sequence and the most closely related sequences found in the database contain a conserved sequence motif, MauG of cytochrome c peroxidases, which is consistent with the oxidative function of RoxA in Xanthomonas sp. RoxA contained approximately 2 mol of heme per mol of protein. This result is in agreement with data for the corresponding gene roxA that postulate the presence of two covalently bound heme molecules per molecule of RoxA . Experiments to extract heme with solvents (acid ethyl acetate or acid methyl ethyl ketone) from purified RoxA were not successful (unpublished observations), confirming the covalent binding of heme to the protein. The inhibition of RoxA by cyanide and carbon monoxide and the shift of the Soret band (406 nm) upon reduction with dithionite (418 nm) or upon incubation with synthetic rubber (409 nm) are in agreement with the involvement of heme in the reaction. Interestingly, addition of catalase did not inhibit RoxA-catalyzed cleavage of NR, suggesting that (free) hydrogen peroxide is not involved in the reaction. The negative results for RoxA in the peroxidase assay are in agreement with the latter finding. Cleavage of polyisoprene by purified RoxA was strictly dependent on the presence of molecular oxygen. In conclusion, RoxA is a novel type of oxygenase. Future experiments will address the function of heme in the reaction mechanism.

ACKNOWLEDGMENTS 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to D.J.

We gratefully acknowledge J. Armbruster (Universität Stuttgart-Hohenheim) for assistance with the HPLC-MS and gas chromatography-MS techniques and for helpful discussions. We also thank M. Priemer and A. Nordheim (Universität Tübingen) for the trypsin fingerprint, matrix-assisted laser desorption ionization—time of flight, and HPLC-MS analyses of RoxA, as well as E. Chua, A. Ikram, and H. Y. Yeang (Rubber Research Institute of Malaysia) for providing purified Hevea latex.