RESULTS

Purification and properties of rubber oxygenase (RoxA).
Xanthomonas sp. produces clearing zones during growth on opaque mineral salts agar with latex as a carbon source. Apparently, Xanthomonas sp. secretes an activity that is able to degrade latex to water-soluble products (Fig. 1A). In order to establish an in vitro assay for latex degradation and to analyze the latex-degrading activity, Xanthomonas sp. was grown on mineral salts medium with purified latex. After 7 to 9 days of incubation the latex was visibly degraded and/or coagulated. The culture fluid was separated from the cells and remaining latex particles by successive centrifugation and filtration (pore size, 0.2 µm). Macromolecular components were concentrated about 20-fold by ultrafiltration (30-kDa cutoff). When aliquots of fresh latex were added to the cell-free concentrated culture fluid and incubated at 30°C, latex was again visibly coagulated or degraded within 2 days; the control, with buffer instead of culture fluid, remained milky. When the same experiment was repeated with either the flowthrough from the 30-kDa filtration step or the concentrated culture fluid that had been heated to 95°C for 10 min before latex was added, no coagulation or degradation was observed. We concluded that the compound responsible for coagulation or degradation of the latex is a heat-sensitive macromolecule, presumably an enzyme. It was noticed that the concentrate had a light reddish color that did not appear to the same extent in glucose-grown cultures. The concentrate had a single absorption maximum around 406 nm (the range from 400 to 600 nm was tested) that could be shifted upon reduction to 418 and 550 nm (data not shown). These results are characteristic for heme-containing proteins, and we speculated that a heme-containing protein was responsible for latex degradation. Using 2 liters of cell-free culture fluid of NR-grown Xanthomonas sp. as the starting material, we were able to purify a protein (apparent molecular mass after sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis, 65 ± 7 kDa) to apparent electrophoretic homogeneity by diafiltration and subsequent chromatography on Q-Sepharose and MonoP (chromatofocusing). The purified 65-kDa protein showed the same strong absorption at 406 nm as the concentrated culture fluid (Fig. 1B and C); the oxidized protein had absorption bands at 280 nm ( band), 356 nm, and 406 nm (Soret band) and weaker and broader bands at 532 nm (ß band), 560 to 565 nm ( band), and 672 nm. A molar extinction coefficient of 1.8 x 10⁵ M^–1 cm^–1 was determined for the absorption at 406 nm, which was similar to the coefficients of diheme enzymes . The ₄₀₆/₂₈₀ value, which reflected the purity and spectral characteristics of RoxA, was 1.17 in 20 mM phosphate buffer (pH 6.8). When the purified 65-kDa protein was reduced by Na₂S₂O₄, absorption, bands appeared at 418 nm (Soret band), 522 nm (ß band), and 549 and 553 nm (both bands), which corresponded to a heme-pyridine complex. These data are indicative of a hemoprotein belonging to the cytochrome c group . Addition of synthetic oligo(1,4-cis-isoprene) to purified RoxA resulted in a shift of the Soret band from 406 to 409 nm, indicating that the substrate binds to the enzyme at the heme site(s). The optical spectra of purified RoxA did not exhibit an absorption band at 695 nm which would be expected for a heme iron-methionine bond.

 

View larger version (44K):   FIG. 1. (A) Clearing zone formation for Xanthomonas sp. after 4 days of incubation at 30°C on opaque latex agar. (B) SDS-polyacrylamide gel electrophoresis of the 65-kDa protein (RoxA) at various stages of purification. Lane M, marker proteins; lane S, concentrated cell-free culture fluid; lane IEX, pool after ion-exchange chromatography on Q-Sepharose; lane IEF, pool after isoelectric focusing on MonoP. (C) Spectra of purified RoxA before (dashed line) and after (solid line) reduction by dithionite. The numbers indicate the observed maxima of the spectra.

 
A trypsin fingerprint analysis of the isolated 65-kDa protein was performed, and the masses of the peptides generated were determined by matrix-assisted laser desorption ionization—time of flight analysis (data not shown). The values obtained for six randomly isolated peptides were in agreement with the values obtained for an in silico trypsin digest of a gene product encoded by a recently cloned gene of Xanthomonas sp. . The cloned gene was assumed to be involved in rubber degradation, but the biochemical function of the protein was not known. Determination of the amino acid sequences of these six peptides confirmed that the 65-kDa protein was identical to the gene product mentioned above (peptide 1, NH₂-YGLYPAPFR; peptide 2, NH₂-TTPITALGNLLPLPWSTGR; peptide 3, NH₂-GLEDEFEDINNFLISLSPATYPK; peptide 4, NH₂-GVAAVVTPIETIR; peptide 5, NH₂-AWNSGWWAYNNLSPSWTGYPSDNIVASELR; peptide 6, NH₂-WALIEYIK). The deduced amino acid sequence of the cloned gene contained two heme-binding motifs (CXXCH) typical for covalently bound heme. Calculation of the heme content of the purified 65-kDa protein by using the molar absorption coefficient of heme-cytochromes (, 29.1 mM^–1 cm^–1 at 551 nm [5]) and three independent methods for protein determination revealed heme and protein concentrations of 16.7 and 9 µM, respectively. These values correspond to a heme content of 1.9 mol of heme per mol of the 65-kDa protein and are in good agreement with the gene sequence analysis that predicted two heme-binding sites. An apparent molecular mass of 54 kDa for the purified native protein was determined by gel filtration (Sephadex G-200). Apparently, the protein has a monomeric subunit structure. In most experiments, the SDS-denatured (reduced) protein migrated at values corresponding to an apparent molecular mass of 55 to 65 kDa, which were significantly lower than the theoretical molecular mass of the mature protein (72.9 kDa) deduced from the gene sequence. The reason for the discrepancy in apparent molecular masses is not known. To test whether the purified protein was responsible for the observed latex-coagulating and -degrading activity, it was added to diluted latex and incubated at 30°C. After incubation for 24 h, controls without enzyme or with boiled enzyme were not changed, but clearing and coagulation of the latex were visible upon incubation with the active enzyme, confirming that the purified 65-kDa protein was responsible for the latex-degrading and -coagulating activity.

Isolation and identification of the main cleavage product of enzymatic rubber degradation.
In order to determine whether the purified protein cleaved the carbon backbone of the polymer or whether it only affected the integrity of the latex emulsion (i.e., the diameter-to-volume ratio of the latex particles), we investigated the presence of low-molecular-mass degradation products in the cleared and coagulated latex. Latex was incubated with the purified 65-kDa protein for several hours at 30°C and subsequently extracted with ethyl acetate; the extracts were analyzed by two-dimensional TLC. One major dark spot became visible when the TLC plate was developed with anisaldehyde, and this spot was absent in controls with either no enzyme or heat-inactivated enzyme (Fig. 2). A few minor low-intensity spots of different color also appeared in some experiments. HPLC analysis of the same ethyl acetate extract revealed only one major peak (at a retention time of 14.8 min) that was missing in control experiments (Fig. 3). The identity of the 14.8-min HPLC fraction that produced the large spot in TLC analysis was established by collecting the corresponding HPLC fraction; subsequent two-dimensional TLC analysis revealed only one spot with the same R_f value (data not shown).

 

View larger version (66K):   FIG. 2. Two-dimensional thin-layer chromatography of degradation products produced from latex by purified RoxA. Latex was allowed to react with purified RoxA before products were extracted with ethyl acetate, dried, and resolved with methanol. Aliquots were spotted onto TLC plates, separated with benzene-acetone (20:1) in the first dimension and with chloroform-methanol (9:1; 90°) in the second dimension, and developed with anisaldehyde-H2SO4 at 100°C.

 

 

View larger version (20K):   FIG. 3. Separation of polyisoprene degradation products by HPLC. Latex was incubated with purified RoxA for 3 h at 40°C and pH 7. Ethyl acetate-extracted products were loaded on a C8 reverse-phase HPLC column as described in Materials and Methods. Heat-inactivated RoxA served as a negative control. mAU, milli absorbance units.

 
The compound which appeared as large dark spot in TLC and as the only prominent peak at 14.8 min in the HPLC analysis thus apparently represented the principal enzymatic rubber degradation product. In a coupled HPLC-MS analysis in the negative ESI mode, the mass spectrum of the 14.8-min fraction showed an [M-H]^– peak at m/z 235 (Fig. 4). When the total ion chromatogram from HPLC was analyzed for additional signals at m/z values that differed from m/z 235 by n isoprene units (i.e., m/z 68), additional [M-H]^– peaks were detected in the m/z 167, 303, 371, and 439 ion chromatograms (Table 1). The levels of these minor metabolites apparently were below the detection limit of the UV diode array in the HPLC analysis. They represent a series of homologous degradation products with one isoprene unit less or one to three isoprene units more than the major metabolite (236 Da).

 

View larger version (9K):   FIG. 4. HPLC-ESI-MS analysis of latex degradation products. Latex degradation products were prepared and separated by HPLC as described in the legend to Fig. 3, and the eluate was monitored by negative ESI-MS. The graph shows the average mass spectrum summed across the 14.8-min peak (seven scans). The relative intensity of the (M+1)-13C isotope peak (m/z 236) corresponds to a C15 molecular formula.

 
The positive ESI mass spectrum of the major metabolite showed an [MH]⁺ peak at m/z 237, as well as the regular adduct ions [M+Na]⁺and [M+K]⁺ and the corresponding cluster ions with methanol (m/z 259, 275, 291, and 307). Two intense peaks at m/z 219 and 201 represented the loss of one or two water molecules from the quasimolecular ion MH⁺, which definitively established the incorporation of two oxygen atoms in the metabolite (data not shown).

When the isolated 236-Da compound was reacted with dinitrophenylhydrazine, a yellow product was obtained, indicating the presence of carbonyl functions in the molecule. Additional structural information was obtained by ¹H-NMR spectroscopy of the isolated compound; the individual resonance signals are shown in Table 2 together with the first-order multiplicity and the corresponding assignments. For the resonance at the lowest field, the chemical shift ( 9.78 ppm), relative intensity 1H, and vicinal coupling constant to the -methylene protons (CH₂, 1.75 Hz) are characteristic of an aldehyde proton. The sharp singlet at 2.14 ppm (relative intensity 3H), on the other hand, is indicative of a nonconjugated acetyl group. Thus, the two ends of the main metabolite can be definitively identified as CHO-CH₂ and CH₂-CO-CH₃, with a combined mass contribution of 100 Da. Since the overall molecular mass is 236 Da, this leaves 136 Da for the core of the metabolite, corresponding to two isoprene moieties [&0807;CH₂-C(CH₃)CH-CH₂; 68 Da each]. The two expected olefinic proton signals for the main metabolite (n = 2) (Fig. 5) were observed at 5.17 and 5.13 ppm. We concluded that 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) (m/z 236) is the formula of the isolated degradation product with a retention time of 14.8 min in HPLC analysis. The experiments described above showed for the first time that in vitro a single enzyme is capable of cleaving the carbon backbone of rubber, yielding 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al as the major degradation product. The purified protein and its corresponding gene were designated rubber oxygenase A (RoxA) and roxA, respectively.

 

View larger version (11K):   FIG. 5. Proposed structure of the latex degradation products produced by RoxA. (A) Molecular structure of the main metabolite at 14.8 min in the HPLC analysis (12-oxo-4,8-dimethyltrideca-4,8-diene-1-al), including the assignments of the 1H-NMR signals (Table 2). (B) General structure of the unequivocally characterized major and minor degradation products, with n = 1 to 5.

 
Biochemical characterization of the NR cleavage reaction with rubber oxygenase (RoxA).
An assay for determination of RoxA activity was developed as follows. The standard assay was performed (see Materials and Methods), and the ethyl acetate-extracted products were separated by HPLC. The RoxA activity was calculated from the peak area at 14.8 min. At low concentrations of RoxA (0.01 to 2 µg/ml of assay mixture) a linear relationship between the area of the 14.8-min peak and the RoxA concentration was found (data not shown). The optimum pH and temperature of purified RoxA were determined to be around pH 7 and 40°C, respectively (Fig. 6). The reaction was strictly dependent on the presence of oxygen, and no rubber degradation product was detected in a nitrogen atmosphere. Addition of potassium cyanide (20 mM) to the assay mixture completely inhibited the reaction. Carbon monoxide also inhibited the reaction if RoxA had been reduced by dithionite before the assay was started in the presence of oxygen (Fig. 7). The results described above are in agreement with the presence of an essential heme(s) in the enzyme. Addition of catalase at various concentrations to the assay system did not affect the reaction at all. Peroxidase activity was not detected even after a prolonged incubation time or if high concentrations of RoxA were used, while a positive control (horseradish peroxidase) reacted within seconds. Alkylation agents, such as N-ethylmaleimide and p-chlorobenzoate, and chelators (bispyridyl, tiron) partially inhibited the reaction. However, EDTA had no significant effect on the activity. Reducing agents and all of the detergents tested (except cholate) strongly inhibited the reaction (Table 3).

 

View larger version (10K):   FIG. 6. Optimum pH (A) and temperature (B) of purified RoxA. Latex was incubated with purified RoxA (1 µg) at different pH values or temperatures for 3 h, and ethyl acetate extracts were separated by HPLC. The area of the peak obtained at 14.8 min was used to calculate the relative amount of degradation product produced. A linear relationship between the amount of RoxA and the peak area was obtained in the range from 10 ng to 2 µg of RoxA per assay mixture. Symbols: •, 0.2 M piperazine-HCl; , 0.2 M bis-Tris-HCl; , 0.2 M Tris-HCl; , 0.2 M ethanolamine-HCl.

 

 

View larger version (19K):   FIG. 7. Dependence of RoxA activity on oxygen and effects of cyanide, carbon monoxide, and catalase. One microgram of purified RoxA was allowed to react with latex in the presence of different compounds. Ethyl acetate extracts were analyzed to determine the area of the peak at 14.8 min by HPLC. Treatments: control (air with 21% oxygen) (RoxA); catalase (1 mM); carbon monoxide (first the atmosphere was replaced by N2 before N2 was replaced by CO, and the assay was subsequently performed under normal air with 21% oxygen); air replaced by N2; addition of 20 mM potassium cyanide.

 
 
RoxA appeared to be specific for oligomers and polymers of 1,4-isoprene. Natural latex and chemosynthetic poly(cis-1,4-isoprene) were significantly cleaved by purified RoxA. When a mixture of chemosynthetic rubber [oligo(cis-1,4-isoprene) with 5 to 15 isoprene units (M_w, 790; M_n, 707; M_w/M_n, 1.12)] was incubated with RoxA, a peak at 14.8 min appeared as a degradation product in HPLC, and the composition of the remaining oligomer mixture was shifted to low-molecular-weight products (data not shown). Oligomers of trans-1,4-isoprene, such as squalene, were hardly cleaved by RoxA, and only small amounts of carbonyl-containing compounds, which were slightly above the detection limit, were detected. When toluene was tested as a substrate, only background activity was obtained. Benzene did not react at all.