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Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp.

ABSTRACT: Biodegradation 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.A 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 (lcp Rr ) 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.Our data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.

SUBMITTER: Watcharakul S

PROVIDER: S-EPMC4877957 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp.

Watcharakul Sirimaporn S Röther Wolf W Birke Jakob J Umsakul Kamontam K Hodgson Brian B Jendrossek Dieter D

BMC microbiology 20160523

<h4>Background</h4>Biodegradation 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.<h4>Results</h4>A rubber-degrading bacterium ...[more]

PMID: 27215318

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Project description:About 22,000 1-methyl-3-nitro-1-nitrosoguanidine- and UV-induced mutants of the rubber-degrading bacterium Streptomyces sp. strain K30 were characterized for the ability to produce clear zones on natural rubber latex overlay agar plates. Thirty-five mutants were defective solely in cleavage of rubber and were phenotypically complemented with the wild-type lcp (latex clearing protein) gene. Sixty-nine mutants exhibited a pleiotropic phenotype and were impaired in utilization of rubber and xylan, indicating that the enzymes responsible for the initial cleavage of these polymers are exported by the same secretion pathway (Q. K. Beg, M. Kapoor, L. Mahajan, and G. S. Hoondal, Appl. Microbiol. Biotechnol. 56:326-3381, 2001; U. K. Laemmli, Nature 227:680-685, 1970). Analysis of the amino acid sequence encoded by lcp revealed a twin-arginine motif, indicating that Lcp is a substrate of the twin-arginine translocation (Tat) pathway (K. Dilks, W. Rose, E. Hartmann, and M. Pohlschröder, J. Bacteriol. 185:1478-1483, 2003). A tatC disruption mutant of Streptomyces lividans 10-164 harboring lcp from Streptomyces sp. strain K30 was not capable of forming clear zones on rubber overlay agar plates. Moreover, Lcp and enhanced green fluorescent protein fusion proteins were detected in the supernatant. Using Escherichia coli having the twin-arginine motif in the signal peptide upstream of Lcp, clear evidence that Lcp is secreted was obtained. Transcriptional analysis revealed basal expression of Lcp in glucose-grown cells and that transcription of lcp is obviously induced in the presence of poly(cis-1,4-isoprene). In contrast, oxiB and oxiA, which are located directly downstream of lcp and putatively encode a heteromultimeric aldehyde dehydrogenase oxidizing the primary cleavage products generated by Lcp from poly(cis-1,4-isoprene), were expressed only in the presence of poly(cis-1,4-isoprene). Expression of lcp at a low level is thus required for sensing the polymer in the medium. Rubber degradation products may then induce the transcription of genes coding for enzymes catalyzing the later steps of poly(cis-1,4-isoprene) degradation and the transcription of lcp itself. lcp, oxiB, and oxiA seem to constitute an operon, as a polycistronic mRNA comprising these three genes was detected. The transcriptional start site of lcp was mapped 400 bp upstream of the lcp start codon.

Project description:Gram-positive rubber degraders such as Streptomyces sp. strain K30 cleave rubber [poly(cis-1,4-isoprene)] to low-molecular-mass oligoisoprenoid products with terminal keto and aldehyde groups by the secretion of a latex clearing protein (Lcp) designated rubber oxygenase. LcpK30 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 LcpK30). Exchange of R164 or T168 by alanine and characterization of the purified LcpK30 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 LcpK30 homologous sequences (H203A, H232A, H259A, H266A). All muteins revealed wild-type properties, excluding any importance for activity and/or heme coordination. Since LcpK30 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 (LcpK30) 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.

Project description: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 ?70 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 C15 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 (?40 kDa), are b-type cytochromes, and cleave polyisoprene to a mixture of C20, C25, C30, 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 C20, C25, C30, 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 water-insoluble 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.

Project description:The latex-clearing protein (Lcp(K30)) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp(K30)), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230- and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp(VH2)) and G. westfalica strain Kb1 (lcp(Kb1)) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to Lcp(K30). Recombinant lcp(VH2) and lcp(Kb1) harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp(VH2) and lcp(Kb1) encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp(VH2) was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-Lcp(K30) immunoglobulin Gs, which were raised in this study, were rather specific for Lcp(K30) and did not cross-react with Lcp(VH2) and Lcp(Kb1). A lcp(VH2) disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp(VH2) seems not to be essential for rubber degradation in G. polyisoprenivorans.

Dataset Information

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp.

Publications

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp.

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