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

本文将比较黄单胞菌二血红素双加氧酶RoxA和链霉菌乳糜清蛋白（Lcp）对聚顺-1,4-异戊二烯橡胶的裂解机理。Lcp是一种非血红素酶，在氨基酸序列上与RoxA无关，但在功能上与RoxA同源。将追求两个目标：(i)我们正在进行的RoxA分析将通过确定氨基酸交换（从RoxA 1.8Å 3D结构分析中选择）对生化（活性，产品组成）和生物物理（UVvis和EPR光谱）特性的影响来继续进行。特别是，我们将确定活性位点附近残基对稳定双氧结合的重要性，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）由于无法从野生型中纯化Lcp和在重组种中异种表达Lcp，过去Lcp的生化分析受到阻碍。最近，我们成功地通过我们的δ - roxa黄单胞菌表达系统以活性形式表达了链霉菌的Lcp，其数量足以用于蛋白分离。我们将纯化Lcp，确定每个Lcp分子金属离子的性质和数量（或将Lcp归类为最近描述的辅酶独立加氧酶组的新成员），我们将确定裂解产物的结构。本文将对Lcp进行全面的生化、生物物理和结构表征，目的是了解两种不相关但独特的细胞外双加氧酶RoxA和Lcp在裂解聚异戊二烯机制中的同源性和差异。

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.