Cleavage of Rubber by the Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30: Molecular Insights

Abstract

Gram-positive rubber degraders such as Streptomyces sp. strain K30 cleave rubber [poly(cis-1,4-isoprene)] to low-molecularmass oligoisoprenoid products with terminal keto and aldehyde groups by the secretion of a latex clearing protein (Lcp) designated rubber oxygenase. Lcp K30 is a heme b cytochrome and has a domain of unknown function (DUF2236) that is characteristic of orthologous Lcps. Proteins with a DUF2236 domain are characterized by three highly conserved residues (R164, T168, and H198 in Lcp K30 ). Exchange of R164 or T168 by alanine and characterization of the purified Lcp K30 muteins revealed that both were stable and contained a heme group (red color) but were inactive. This finding identifies both residues as key residues for the cleavage reaction. The purified H198A mutein was also inactive and stable but was colorless due to the absence of heme. We constructed and characterized alanine muteins of four additional histidine residues moderately conserved in 495 Lcp K30 homologous sequences (H203A, H232A, H259A, H266A). All muteins revealed wild-type properties, excluding any importance for activity and/or heme coordination. Since Lcp K30 has only eight histidines and the three remaining residues (H103, H184, and H296) were not conserved (<11%), H198 presumably is the only essential histidine, indicating its putative function as a heme ligand. The second axial position of the heme is likely occupied by a not yet identified molecule. Mutational analysis of three strictly conserved arginine residues (R195, R202, R328) showed that R195A and R202A muteins were colorless and instable, suggesting that these residues are important for the protein stability. IMPORTANCELarge amounts of rubber waste materials have been permanently released into the environment for more than a century, yet accumulation of rubber particles released, e.g., by abrasion of tires along highways has not been observed. This is indicative of the ubiquitous presence and activity of rubber-degrading microorganisms. Despite increasing research activities on rubber biodegradation during the last 2 decades, the knowledge of the enzymatic cleavage mechanism of rubber by latex clearing protein (Lcp) still is limited. In particular, the catalytic cleavage mechanism and the amino acids of Lcp proteins (Lcps) that are involved have not yet been identified for any Lcp. In this study, we investigated the importance of 10 amino acid residues of Lcp from Streptomyces sp. K30 (Lcp K30 ) by mutagenesis, mutein purification, and biochemical characterization. We identified several essential residues, one of which most likely represents an axial heme ligand in Lcp of Streptomyces sp. K30. Microbial degradation of poly(cis-1,4-isoprene) depends on rubber oxygenases that are synthesized by rubber-degrading microorganisms. Two types of rubber oxygenases are currently known: one is rubber oxygenase RoxA that has been studied in Xanthomonas sp. strain 35Y (1). RoxA is a c-type diheme-dioxygenase and has been found only in Gram-negative rubber degraders...

Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber

“…Site-directed mutagenesis of the lcp K30 gene and subsequent cloning and expression of the mutant lcp K30 gene was performed by the QuikChange method as described in detail previously 26 using oligonucleotides shown in Suppl. Fig.…”

Production of functionalized oligo‐isoprenoids by enzymatic cleavage of rubber

“…35Y strains which harboured either the roxA Xsp or the roxB Xsp gene integrated into the chromosome under the control of an L‐rhamnose‐inducible promoter using a two‐step purification procedure as described recently (Birke et al ., , ). Lcp K30 was expressed intracellularly in form of an N‐terminal Strep‐tagged protein and was purified from recombinant E. coli as described previously (Röther et al ., ). Fig.…”

“…Lcps are b ‐type cytochromes and share a common domain of unknown function (DUF2236) (Hiessl et al ., ; Birke et al ., ). Recently, the importance of several strictly conserved residues within the DUF2236 domain for stability and activity was determined (Röther et al ., ).…”

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

“…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. Moreover, Lcps differ from RoxAs in having only one noncovalently bound heme (b-type cytochromes) and produce a mixture of C 20 , C 25 , C 30 , and higher oligo-isoprenoids as end products.…”