Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23°C and 37°C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1 VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in Lcp K30 . Heme was identified as a cofactor in purified Lcp K30 by (i) detection of characteristic ␣-, -, and ␥ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetoneextractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe 3؉ that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. Lcp K30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5°C (RoxA, 54.3°C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms.T he hydrocarbon natural rubber [poly(cis-1,4-isoprene)] is an important biopolymer that is produced by many plants and some fungi. Natural rubber, as well as chemically synthesized poly(cis-1,4-isoprene) (synthetic rubber), has been in use by mankind for more than 100 years. Huge amounts of rubber derived from the rubber tree Hevea brasiliensis are the basis for the manufacturing of tires, sealers, latex gloves, condoms, and many other items. Most of these materials are released to the environment in the form of waste or by abrasion in the case of tires. As a natural compound, rubber is a fully biodegradable material, and many rubber-degrading microorganisms have been isolated from various ecosystems in the past (1-8). The initial step of rubber biodegradation is the enzymatic cleavage of the polymeric carbon backbone to smaller products. Two types of rubber-cleaving enzymes have been characterized so far. One is rubber oxygenase A, RoxA, which is found in Gram-negative clearing zone formers. The other is latex clearing protein, Lcp, which has been identified only in Gram-positive organisms so far. RoxA first was isolated and biochemically characterized from Xanthomonas sp. strain 35Y (9). Biochemical and biophysical investigation revealed that RoxA is an extracellular dioxygenase with two covalently attached heme groups (10, 11) and is structurally but not functio...
BackgroundBiodegradation of rubber (polyisoprene) is initiated by oxidative cleavage of the polyisoprene backbone and is performed either by an extracellular rubber oxygenase (RoxA) from Gram-negative rubber degrading bacteria or by a latex clearing protein (Lcp) secreted by Gram-positive rubber degrading bacteria. Only little is known on the biochemistry of polyisoprene cleavage by Lcp and on the types and functions of the involved cofactors.ResultsA rubber-degrading bacterium was isolated from the effluent of a rubber-processing factory and was taxonomically identified as a Rhodococcus rhodochrous species. A gene of R. rhodochrous RPK1 that coded for a polyisoprene-cleaving latex clearing protein (lcpRr) was identified, cloned, expressed in Escherichia coli and purified. Purified LcpRr had a specific activity of 3.1 U/mg at 30 °C and degraded poly(1,4-cis-isoprene) to a mixture of oligoisoprene molecules with terminal keto and aldehyde groups. The pH optimum of LcpRr was higher (pH 8) than for other rubber-cleaving enzymes (≈ pH 7). UVvis spectroscopic analysis of LcpRr revealed a cytochrome-specific absorption spectrum with an additional feature at long wavelengths that has not been observed for any other rubber-cleaving enzyme. The presence of one b-type haem in LcpRr as a co-factor was confirmed by (i) metal analysis, (ii) solvent extraction, (iii) bipyridyl assay and (iv) detection of haem-b specific m/z values via mass-spectrometry.ConclusionsOur data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0703-x) contains supplementary material, which is available to authorized users.
Latex clearing proteins (Lcps) are rubber oxygenases that catalyse the extracellular cleavage of poly (cis-1,4-isoprene) by Gram-positive rubber degrading bacteria. Lcp of Streptomyces sp. K30 (LcpK30) is a b-type cytochrome and acts as an endo-type dioxygenase producing C20 and higher oligo-isoprenoids that differ in the number of isoprene units but have the same terminal functions, CHO-CH2– and –CH2-COCH3. Our analysis of the LcpK30 structure revealed a 3/3 globin fold with additional domains at the N- and C-termini and similarities to globin-coupled sensor proteins. The haem group of LcpK30 is ligated to the polypeptide by a proximal histidine (His198) and by a lysine residue (Lys167) as the distal axial ligand. The comparison of LcpK30 structures in a closed and in an open state as well as spectroscopic and biochemical analysis of wild type and LcpK30 muteins provided insights into the action of the enzyme during catalysis.
Gram-positive rubber degraders such as Streptomyces sp. strain K30 cleave rubber [poly(cis-1,4-isoprene)] to low-molecularmass oligoisoprenoid products with terminal keto and aldehyde groups by the secretion of a latex clearing protein (Lcp) designated rubber oxygenase. Lcp K30 is a heme b cytochrome and has a domain of unknown function (DUF2236) that is characteristic of orthologous Lcps. Proteins with a DUF2236 domain are characterized by three highly conserved residues (R164, T168, and H198 in Lcp K30 ). Exchange of R164 or T168 by alanine and characterization of the purified Lcp K30 muteins revealed that both were stable and contained a heme group (red color) but were inactive. This finding identifies both residues as key residues for the cleavage reaction. The purified H198A mutein was also inactive and stable but was colorless due to the absence of heme. We constructed and characterized alanine muteins of four additional histidine residues moderately conserved in 495 Lcp K30 homologous sequences (H203A, H232A, H259A, H266A). All muteins revealed wild-type properties, excluding any importance for activity and/or heme coordination. Since Lcp K30 has only eight histidines and the three remaining residues (H103, H184, and H296) were not conserved (<11%), H198 presumably is the only essential histidine, indicating its putative function as a heme ligand. The second axial position of the heme is likely occupied by a not yet identified molecule. Mutational analysis of three strictly conserved arginine residues (R195, R202, R328) showed that R195A and R202A muteins were colorless and instable, suggesting that these residues are important for the protein stability. IMPORTANCELarge amounts of rubber waste materials have been permanently released into the environment for more than a century, yet accumulation of rubber particles released, e.g., by abrasion of tires along highways has not been observed. This is indicative of the ubiquitous presence and activity of rubber-degrading microorganisms. Despite increasing research activities on rubber biodegradation during the last 2 decades, the knowledge of the enzymatic cleavage mechanism of rubber by latex clearing protein (Lcp) still is limited. In particular, the catalytic cleavage mechanism and the amino acids of Lcp proteins (Lcps) that are involved have not yet been identified for any Lcp. In this study, we investigated the importance of 10 amino acid residues of Lcp from Streptomyces sp. K30 (Lcp K30 ) by mutagenesis, mutein purification, and biochemical characterization. We identified several essential residues, one of which most likely represents an axial heme ligand in Lcp of Streptomyces sp. K30. Microbial degradation of poly(cis-1,4-isoprene) depends on rubber oxygenases that are synthesized by rubber-degrading microorganisms. Two types of rubber oxygenases are currently known: one is rubber oxygenase RoxA that has been studied in Xanthomonas sp. strain 35Y (1). RoxA is a c-type diheme-dioxygenase and has been found only in Gram-negative rubber degraders...
Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are c-type cytochromes of Ϸ70 kDa produced by Gram-negative rubber-degrading bacteria, and they cleave polyisoprene into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD), a C 15 oligo-isoprenoid, as the major end product. Lcps are common among Gram-positive rubber degraders and do not share amino acid sequence similarities with RoxAs. Furthermore, Lcps have much smaller molecular masses (Ϸ40 kDa), are b-type cytochromes, and cleave polyisoprene to a mixture of C 20 , C 25 , C 30 , and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB Xsp (RoxB of Xanthomonas sp. strain 35Y). RoxB Xsp is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c-type hemes). However, RoxB Xsp differs from all currently known RoxAs in having a distinctive product spectrum of C 20 , C 25 , C 30 , and higher oligo-isoprenoids that has been observed only for Lcps so far. Purified RoxB Xsp revealed the highest specific activity of 4.5 U/mg (at 23°C) of all currently known rubber oxygenases and exerts a synergistic effect on the efficiency of polyisoprene cleavage by RoxA Xsp . RoxB homologs were identified in several other Gram-negative rubber-degrading species, pointing to a prominent function of RoxB for the biodegradation of rubber in Gram-negative bacteria.IMPORTANCE The enzymatic cleavage of rubber (polyisoprene) is of high environmental importance given that enormous amounts of rubber waste materials are permanently released (e.g., by abrasion of tires). Research from the last decade has discovered rubber oxygenase A, RoxA, and latex clearing protein (Lcp) as being responsible for the primary enzymatic attack on the hydrophobic and waterinsoluble biopolymer poly(cis-1,4-isoprene) in Gram-negative and Gram-positive rubber-degrading bacteria, respectively. Here, we provide evidence that a third type of rubber oxygenase is present in Gram-negative rubber-degrading species. Due to its characteristics, we suggest the designation RoxB for the new type of rubber oxygenase. Bioinformatic analysis of genome sequences indicates the presence of roxB homologs in other Gram-negative rubber degraders.KEYWORDS rubber oxygenase, latex clearing protein, polyisoprene, biodegradation, heme dioxygenase, dioxygenases M aterials that contain or consist completely of rubber [poly(cis-1,4-isoprene)] have been in use by mankind for more than 100 years at an industrial scale. Most of the rubber materials are released into the environment after use. In particular, small rubber particles liberated from car tires by abrasion contribute to a constant supply of rubber to many ecosystems on earth. Therefore, it is not astonishing that rubber-degrading microorganisms are widely distributed, and several research reports have described the
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