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 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...

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

“…Figure 5B shows the product profiles obtained for RoxB Xsp , RoxA Xsp , and Lcp K30 . To our surprise, the identified products of the RoxB Xsp -catalyzed reaction were the same as those previously determined for Lcp K30 (21) or for Lcp Rr from Rhodococcus rhodochrous (22). In all cases, a mixture of C 20 , C 25 , and higher oligo-isoprenoids with terminal aldehyde and keto groups were identified.…”

“…Carbon monoxide is able to bind to RoxA Xsp in the form as isolated, as revealed by the CO-dependent appearance of an absorption peak at 415 nm in the difference spectrum (21). This binding can be explained by the CO affinity of the reduced, oxygen-bearing heme center (10).…”

“…This binding can be explained by the CO affinity of the reduced, oxygen-bearing heme center (10). Carbon monoxide strongly inhibits the polyisoprene cleavage reaction of RoxA Xsp (21). When we incubated purified RoxB Xsp as isolated with carbon monoxide, no change in the UV-vis spectrum was detected.…”

“…Purifications of Lcp K30 and RoxA Xsp were performed as described previously (9,21). RoxB Xsp was purified from the supernatant of a Xanthomonas sp.…”

“…Investigation of rubber degradation by Gram-positive species in A. Steinbüchel=s group led to the identification of a second type of rubber oxygenase (named latex clearing protein [Lcp]) (13)(14)(15)(16)(17)(18). Meanwhile the Lcps from species of four different genera (Streptomyces, Gordonia, Rhodococcus, and Nocardia) have been purified and biochemically characterized (19)(20)(21)(22)(23). Lcps from different species are related in amino acid sequence and share a domain of unknown function, DUF2236 (19,24), but differ from RoxAs in several properties: the molecular masses of Lcps are only approximately half (Ϸ40 kDa) of that of RoxAs (Ϸ75 kDa), and Lcps don't share amino acid similarities to RoxAs.…”