Novel Type of Heme-Dependent Oxygenase Catalyzes Oxidative Cleavage of Rubber (Poly-cis-1,4-Isoprene)

Abstract

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latexgrown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH 2 O and OCH 2 -COCH 3 . An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly ( Natural rubber (NR) is a biopolymer that is synthesized by many plants and some fungi. This polymer has been commercially exploited for more than 100 years by cultivating and tapping the rubber tree (Hevea brasiliensis). NR is a polymer of many isoprene units [poly(cis-1,4-isoprene)] that, after cross-linking of the linear polymer chains by sulfur bridges (vulcanization), has superior physical properties. Despite the development of chemosynthetic rubbers, NR is still a necessary raw material for products such as tires, latex gloves, condoms, and seals.NR does not accumulate in the environment. Many reports on the biodegradability of rubbers were published during the last century (for recent studies see references 2, 9, 11, 12, and 18 and references therein). Even chemically cross-linked (vulcanized) rubbers have been shown to be biodegradable (3,7,17). Two biological strategies for microbial NR degradation have been described so far. (i) A large number of bacteria, most of which belong to the actinomycetes, are able to grow and to produce clearing zones on agar media containing NR latex in the form of a milky opaque emulsion as a carbon source (9). So far, Xanthomonas sp. strain 35Y is the only known gram-negative NR-degrading bacterium belonging to this group (18). (ii) The members of the other group of NR-utilizing bacteria do not produce clearing zones on NR latex agar; rather, they are able to solubilize solid pieces of NR and to use the resulting emulsion as a carbon sou...

Crystallization of the extracellular rubber oxygenase RoxA fromXanthomonassp. strain 35Y

“…strain 35Y and purification of the extracellular protein RoxA from the cell-free culture supernatant were performed as described previously (Braaz et al, 2004(Braaz et al, , 2005. Approximately 5 mg of protein obtained in a single purification were concentrated to 4 mg ml À1 by ultrafiltration and exchanged into a buffer containing 5 mM Tris-HCl pH 8.5 by size-exclusion chromatography on Sephadex G25.…”

“…The protein was purified to apparent electrophoretic homogeneity (Braaz et al, 2004). Crystals of RoxA were obtained by sitting-drop vapour diffusion at 293 K (Fig.…”

“…The cleavage of poly(cis-1,4-isoprene) by purified RoxA is strictly dependent on the presence of molecular oxygen (Braaz et al, 2004(Braaz et al, , 2005. To date, the basic molecular mechanism by which poly(cis-1,4-isoprene) is degraded is not known.…”

“…strain 35Y and showed a strong absorption maximum at 406 nm (Braaz et al, 2004). The amino-acid sequence of RoxA comprises two haem-binding motifs Cys-X 1 -X 2 -Cys-His, which are typical attachment sites for covalently bound haem; RoxA was thus classified as a dihaem c-type cytochrome (Jendrossek & Reinhardt, 2003).…”

“…During growth on poly(cis-1,4-isoprene), Xanthomonas sp. strain 35Y secretes a haem-containing protein, rubber oxygenase A (RoxA), into the medium (Braaz et al, 2004). Purified RoxA was shown to cleave poly(cis-1,4-isoprene) in vitro by incorporation of oxygen to yield as the major cleavage product, with a series of related minor byproducts differing only in the number of isoprene units (Braaz et al, 2004;Fig.…”