Abstract
Latex clearing proteins (Lcps) are rubber oxygenases that catalyse the extracellular cleavage of poly (cis-1,4-isoprene) by Gram-positive rubber degrading bacteria. Lcp of Streptomyces sp. K30 (LcpK30) is a b-type cytochrome and acts as an endo-type dioxygenase producing C20 and higher oligo-isoprenoids that differ in the number of isoprene units but have the same terminal functions, CHO-CH2– and –CH2-COCH3. Our analysis of the LcpK30 structure revealed a 3/3 globin fold with additional domains at the N- and C-termini and similarities to globin-coupled sensor proteins. The haem group of LcpK30 is ligated to the polypeptide by a proximal histidine (His198) and by a lysine residue (Lys167) as the distal axial ligand. The comparison of LcpK30 structures in a closed and in an open state as well as spectroscopic and biochemical analysis of wild type and LcpK30 muteins provided insights into the action of the enzyme during catalysis.
Highlights
Despite the economic importance of natural rubber and the considerable quantities of rubber waste that are permanently released into the environment, the knowledge about the fate of rubber materials in nature is still limited
Crystals of latex clearing protein (Lcp) from Streptomyces sp. K30 (LcpK30) belong to the triclinic space group P1 with two monomers
We provide evidence that the distal haem site with Arg[164] and Thr[168] in the vicinity of the distal haem position Lys[167] represents the active site of LcpK30
Summary
Despite the economic importance of natural rubber and the considerable quantities of rubber waste that are permanently released into the environment, the knowledge about the fate of rubber materials in nature is still limited. K30 (lcpK30)[18] many other lcp-like genes were described, for instance in rubber-degrading Gordonia species (G. polyisoprenivorans, G. westfalica)[6, 19, 20], in Rhodococcus rhodochrous RPK1 (lcpRr)[21] and recently in other Streptomyces species[8]. The amino acid sequences of RoxA and Lcp largely differ in length and show no relevant similarity, but despite this dissimilarity both enzyme types have a monomeric quaternary structure and catalyse the oxidative cleavage of the double bonds in poly (cis-1,4-isoprene) and produce similar cleavage products with terminal keto and aldehyde groups. In contrast to RoxAs that cleave rubber in an endo-type, processive manner to a single major end product (ODTD, C15 oligoisoprenoid), Lcps produce a mixture of cleavage products that differ in the number of central isoprene units[24, 27]. We provide insights into the function of the active site obtained by spectroscopic methods and by the evaluation of previously published and newly generated results from mutagenesis of amino acid residues close to the active site
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