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

摘要

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 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. 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 , 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.