Cleavage of Rubber by the Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30: Molecular Insights
- 15 November 2016
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 82 (22), 6593-6602
- https://doi.org/10.1128/aem.02176-16
Abstract
Gram-positive rubber degraders such as Streptomyces sp. strain K30 cleave rubber [poly( cis -1,4-isoprene)] to low-molecular-mass 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 (IMPORTANCE Large 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.Keywords
Funding Information
- Deutsche Forschungsgemeinschaft
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