title: Latex clearing protein-an oxygenase cleaving poly(cis-1,4-isoprene) rubber at the cis double bonds.
abstract: Gordonia polyisoprenivorans strain VH2, a potent rubber-degrading actinomycete, harbors two latex clearing proteins (Lcps), which are known to be essential for the microbial degradation of rubber. However, biochemical information on the exact role of this protein in the degradation of polyisoprene was lacking. In this study, the gene encoding Lcp1VH2 was heterologously expressed in strains of Escherichia coli, the corresponding protein was purified, and its role in rubber degradation was examined by measurement of oxygen consumption as well as by chromatographic and spectroscopic methods. It turned out that active Lcp1VH2 is a monomer and is responsible for the oxidative cleavage of poly(cis-1,4-isoprene) in synthetic as well as in natural rubber by the addition of oxygen (O2) to the cis double bonds. The resulting oligomers possess repetitive isoprene units with aldehyde (CHO-CH2-) and ketone (-CH2-CO-CH3) functional groups at the termini. Two fractions with average isoprene contents of 18 and 10, respectively, were isolated, thus indicating an endocleavage mechanism. The activity of Lcp1VH2 was determined by applying a polarographic assay. Alkenes, acyclic terpenes, or other rubber-like polymers, such as poly(cis-1,4-butadiene) or poly(trans-1,4-isoprene), are not oxidatively cleaved by Lcp1VH2. The pH and temperature optima of the enzyme are at pH 7 and 30°C, respectively. Furthermore, it was demonstrated that active Lcp1VH2 is a Cu(II)-containing oxygenase that exhibits a conserved domain of unknown function which cannot be detected in any other hitherto-characterized enzyme. The results presented here indicate that this domain might represent a new protein family of oxygenases.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hiessl
authors.affiliation: Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Dietrich
authors.initials: D
authors.lastname: Böse
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Oetermann
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Eggers
authors.affiliation: Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, Jülich, Germany Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Pietruszka
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany Environmental Sciences Department, King Abdulaziz University, Jeddah, Saudi Arabia steinbu@uni-muenster.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinbüchel
mesh_terms: D001711:Biotransformation; D002845:Chromatography; D003067:Coenzymes / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D033601:Gordonia Bacterium / Q000201:enzymology*; D045782:Hemiterpenes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007840:Latex / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011048:Polarography; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012408:Rubber / Q000378:metabolism*; D013057:Spectrum Analysis; D013696:Temperature
chemical_list: D003067:Coenzymes; D045782:Hemiterpenes; D007840:Latex; D011994:Recombinant Proteins; D003300:Copper; C455100:1,4-polyisoprene; D012408:Rubber; D010105:Oxygenases; D010100:Oxygen