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

本文对黄单胞菌的二血红素双加氧酶RoxA和链霉菌的胶乳透明蛋白（LCP）对聚（顺式-1，4-异戊二烯）（橡胶）的裂解机理进行了测定和比较。LCP是一种非血红素酶，氨基酸序列上与RoxA无关，但功能上与RoxA同源。将追求两个目标：（i）我们正在进行的RoxA分析将继续通过确定氨基酸交换（选自RoxA 1.8 μ m 3D结构分析）对生物化学（活性，产物组成）和生物物理（UVvis和EPR光谱）特性的影响。特别是，我们将确定活性位点附近的残基对稳定分子氧结合的重要性，并将确定形成（疏水）底物通道的氨基酸，并构成负责形成确定长度的裂解产物（C15-化合物）的分子标尺。(ii)由于不能从野生型中纯化Lcp和在重组物种中异源表达Lcp，过去Lcp的生化分析受到阻碍。最近，我们成功地表达LCP的链霉菌。在活性形式通过我们的delta-roxA黄单胞菌属的表达系统的数量足够的蛋白质分离。我们将纯化LCP，确定每个分子LCP的假定金属离子的性质和数量（或将LCP分类为最近描述的非辅因子依赖性加氧酶组的新成员），我们将确定裂解产物的结构。一个全面的生化，生物物理和结构表征的LCP将进行的目的是了解的同源性和差异的裂解机制的聚异戊二烯由两个不相关的，但独特的类型的细胞外双加氧酶，RoxA和LCP。

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.