title: 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.
abstract: 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 Lcp1VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in LcpK30. Heme was identified as a cofactor in purified LcpK30 by (i) detection of characteristic α-, β-, and γ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetone-extractable 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. LcpK30 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.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Germany.
authors.forename: Jakob
authors.initials: J
authors.lastname: Birke
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Germany.
authors.forename: Wolf
authors.initials: W
authors.lastname: Röther
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Germany dieter.jendrossek@imb.uni-stuttgart.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D055592:Biophysical Phenomena; D003067:Coenzymes / Q000032:analysis; D045303:Cytochromes b / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000032:analysis; D007840:Latex / Q000378:metabolism*; D008670:Metals / Q000032:analysis; D010105:Oxygenases / Q000378:metabolism*; D013057:Spectrum Analysis; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature; D044366:Transition Temperature
chemical_list: D003067:Coenzymes; D007840:Latex; D008670:Metals; D006418:Heme; D045303:Cytochromes b; D010105:Oxygenases
references.citation: Anal Biochem. 1965 Jul;12(1):77-82
references.pmid: 4285061
references.citation: Appl Environ Microbiol. 2008 Sep;74(17):5373-82
references.pmid: 18606806
references.citation: Appl Environ Microbiol. 2013 Oct;79(20):6391-9
references.pmid: 23934498
references.citation: Appl Environ Microbiol. 2004 Dec;70(12):7388-95
references.pmid: 15574940
references.citation: Appl Environ Microbiol. 2005 May;71(5):2473-8
references.pmid: 15870336
references.citation: FEMS Microbiol Lett. 1997 May 15;150(2):179-88
references.pmid: 9170260
references.citation: Biochim Biophys Acta. 1999 May 5;1411(2-3):290-309
references.pmid: 10320664
references.citation: Appl Environ Microbiol. 2012 Apr;78(8):2874-87
references.pmid: 22327575
references.citation: J Gen Appl Microbiol. 2013;59(3):199-205
references.pmid: 23863290
references.citation: Appl Environ Microbiol. 2014 Aug;80(16):5012-20
references.pmid: 24907333
references.citation: Appl Environ Microbiol. 1995 Aug;61(8):3092-7
references.pmid: 16535106
references.citation: Microbiologyopen. 2012 Mar;1(1):13-24
references.pmid: 22950008
references.citation: Appl Environ Microbiol. 2014 Sep;80(17):5231-40
references.pmid: 24928880
references.citation: Appl Environ Microbiol. 2000 Apr;66(4):1639-45
references.pmid: 10742254
references.citation: J Appl Microbiol. 2010 Sep;109(3):1067-75
references.pmid: 20408935
references.citation: Appl Environ Microbiol. 2012 Jul;78(13):4543-51
references.pmid: 22504822
references.citation: Appl Environ Microbiol. 2005 Jun;71(6):2803-12
references.pmid: 15932971
references.citation: Biochemistry. 2005 Nov 22;44(46):15406-13
references.pmid: 16285745
references.citation: Microbiology. 2010 Aug;156(Pt 8):2537-48
references.pmid: 20413555
references.citation: Biomacromolecules. 2005 Jan-Feb;6(1):180-8
references.pmid: 15638519
references.citation: Appl Environ Microbiol. 2012 Nov;78(22):7876-83
references.pmid: 22941074
references.citation: Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13833-8
references.pmid: 23922395
references.citation: J Biol Chem. 1977 Jul 10;252(13):4637-42
references.pmid: 194886
references.citation: Anal Biochem. 1987 Feb 15;161(1):1-15
references.pmid: 3578775
references.citation: Enzyme Microb Technol. 2011 Dec 10;49(6-7):526-31
references.pmid: 22142727