Novel type of heme-dependent oxygenase catalyzes oxidative cleavage of rubber (poly-cis-1,4-isoprene)

Appl Environ Microbiol. 2004 Dec;70(12):7388-95. doi: 10.1128/AEM.70.12.7388-7395.2004.

Abstract

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2--and--H2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40 degrees C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Butadienes / metabolism*
  • Heme Oxygenase (Decyclizing) / chemistry
  • Heme Oxygenase (Decyclizing) / genetics
  • Heme Oxygenase (Decyclizing) / isolation & purification
  • Heme Oxygenase (Decyclizing) / metabolism*
  • Hemiterpenes / metabolism*
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Pentanes / metabolism*
  • Rubber / metabolism*
  • Xanthomonas / enzymology*
  • Xanthomonas / genetics
  • Xanthomonas / growth & development

Substances

  • Bacterial Proteins
  • Butadienes
  • Hemiterpenes
  • Pentanes
  • isoprene
  • Rubber
  • Heme Oxygenase (Decyclizing)