Mechanisms of enzymatic polyisoprene cleavage by rubber oxygenase RoxA and latex clearing protein LCP

• Applicant: Professor Dr. Dieter Jendrossek
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2011 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 194272362

The cleavage mechanisms of poly(cis-1,4-isoprene) (rubber) by diheme dioxygenase RoxA of Xanthomonas sp. and by latex clearing protein (Lcp) of Streptomyces sp. will be determined and compared. Lcp is a non-heme enzyme unrelated in amino acid sequence but homologous to RoxA in function. Two objectives will be pursued: (i) our ongoing analysis of RoxA will be continued by determination of the effect of amino acid exchanges (selected from analysis of the RoxA 1.8Å 3D structure) on biochemical (activity, product composition) and biophysical (UVvis and EPR spectra) properties. In particular, we will determine the importance of residues near the active site for stabilization of dioxygen binding and will identify amino acids that form a (hydrophobic) substrate channel and constitute a molecular ruler responsible for formation of a cleavage product of defined length (C15-compound). (ii) Biochemical analysis of Lcp in the past was hampered by the failure to purify Lcp from wild types and to heterologically express Lcp in recombinant species. Recently, we succeeded to express Lcp of Streptomyces sp. in active form via our delta-roxA Xanthomonas sp. expression system in quantities sufficient for protein isolation. We will purify Lcp, determine the nature and number of postulated metal ions per molecule Lcp (or classify Lcp as a new member of the recently described group of cofactor-independent oxygenases) and we will identify the structure of cleavage products. A comprehensive biochemical, biophysical and structural characterization of Lcp will be performed with the aim to understand the homologies and differences in the cleavage mechanism of polyisoprene by two unrelated but unique types of extracellular dioxygenases, RoxA and Lcp.

• DFG Programme: Research Grants