#Carbon backbone series#

The reaction mixture was incubated at 40☌ for 3 h, and the degradation products were extracted and analyzed as described above. The two anaerobic samples were combined under anaerobic conditions, and 18O 2 was added to a final concentration of about 20% (vol/vol). Oxygen was removed by repeated evacuation and flushing with nitrogen gas. For the 18O 2-labeling experiment, rubber latex (4 μl of a 35% emulsion) in bis-Tris buffer (896 μl, 200 mM, pH 7.0) and purified RoxA (100 μl, 10 to 15 μg/ml) were transferred into separate 10-ml glass tubes and sealed with rubber stoppers. Isotope-labeling experiments were performed using 18O 2 (97% Sigma-Aldrich, Schnelldorf, Germany) and H 2 18O (95% Euriso-top, Saarbrücken, Germany). These fractions were pooled and passed through a Superdex 200 column (Superdex 200 Prep-grade Pharmacia) and eluted with phosphate buffer (20 mM, pH = 7.0). During elution with a linear phosphate buffer gradient up to 500 mM fractions with the characteristic spectrum of RoxA were eluted at approximately 20 mM. Fractions showing the characteristic absorption spectrum of RoxA ( 2) were pooled and, after desalting and changing the buffer by diafiltration (30-kDa cutoff), applied to a hydroxyapatite column (CHT 5-I Bio-Rad) preequilibrated with 5 mM phosphate buffer (pH = 6.8) at a flow rate of 2.0 ml min −1. RoxA was eluted from the column with a linear gradient of 0 to 0.5 M NaCl in basic buffer at approximately 250 mM. cells was concentrated by ultrafiltration (30-kDa cutoff) and passed through a Q-Sepharose column (HP HR16/10 Pharmacia) preequilibrated with basic buffer (20 mM 1,3-diaminopropane/HCl ) at a flow rate of 1 ml min −1. Cell-free supernatant of latex-grown Xanthomonas sp. The enzyme was purified at 5☌ using a fast-performance liquid chromatography system consisting of an LCC 500 controller, a 500 pump, a UV-1 monitor, a Rec-482 recorder, and a FRAC autosampler (Pharmacia, Uppsala, Sweden). MATERIALS AND METHODS Bacteria, media, and culture conditions. In this contribution, we continued our studies on RoxA and analyzed the oxidative cleavage reaction of RoxA by 18O-labeling experiments.

#Carbon backbone series#

Purified RoxA was able to cleave poly( cis-1,4-isoprene) in vitro to 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD) as the major cleavage product and a series of related low-molecular-weight products differing from ODTD only by the number of isoprene units as minor metabolites ( 2).

and characterized its gene (rubber oxygenase, roxA) ( 5). We purified an extracellular heme-containing protein from latex-grown culture fluid of Xanthomonas sp. This bacterium produces translucent clearing zones during growth on solid media containing opaque latex fluid as the carbon source ( 12). Recently, we initiated analysis of rubber degradation by Xanthomonas sp. Many reports on the isolation and characterization of rubber-degrading microorganisms have been published ( 4, 6, 8, 12, and references cited therein), but the basic molecular mechanism by which poly( cis-1,4-isoprene) is degraded is unknown. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly( cis-1,4-isoprene) is proposed. In the presence of 18O 2, H 2 16O, and alcohol dehydrogenase/NADH, incorporation of two atoms of 18O into the reduced metabolite HDTD was found ( m/ z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD m/ z 240) during RoxA-mediated in vitro cleavage of poly( cis-1,4-isoprene). Incubation of poly( cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H 2 18O in the presence of 16O 2 indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. Incorporation of one 18O atom in ODTD was found if the cleavage reaction was performed in the presence of 18O 2 and H 2 16O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD m/ z 236) was the main cleavage product in the absence of 18O-compounds. was investigated in the presence of different combinations of 16O 2, 18O 2, H 2 16O, and H 2 18O. Oxidative cleavage of poly( cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp.