Development of a homologous expression system for rubber oxygenase RoxA from Xanthomonas sp.

Hambsch

Schmitt

et al. 2012

RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (Ϸ5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ⌬roxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to Ϸ3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.

“…In contrast to previous assumptions (6), expression of recombinant roxA in Xanthomonas sp. from plasmids provided in trans was not possible.…”

Section: Methodscontrasting

confidence: 41%

Section: Resultsmentioning

confidence: 76%

See 1 more Smart Citation

Phe317 Is Essential for Rubber Oxygenase RoxA Activity

Hambsch

Schmitt

et al. 2012

“…Attempts to express roxB Xsp in Escherichia coli were not successful (not shown). The growth-inhibitory effect of plasmid-derived expression of roxA in E. coli has been described previously (11,28). We therefore integrated roxB Xsp under the control of an L-rhamnose-inducible promoter into the genome of a ΔroxA background of Xanthomonas sp.…”

Section: Resultsmentioning

confidence: 99%

RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp)

Röther

Jendrossek

2017

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

“…RoxA was purified from a ⌬roxA Xanthomonas sp. with chromosomally integrated roxA plasmid (pNH1::roxA) under rhamnose control as described previously (12,21). Lcp with the replacement of the TAT sequence by a Strep-tag was purified by using E. coli JM109 harboring p4782.1::lcp (volume, 4.8 liters) grown at 22°C for 20 h. The cells were harvested and resuspended in buffer A (1 ml buffer A/g cells, 100 mM potassium phosphate buffer [KPP], pH 7.7, 150 mM sodium chloride).…”

Section: Methodsmentioning

confidence: 99%

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b -Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

Röther

Jendrossek

2015

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...