Lehrstuhl für Biochemie, Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany.
Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13833-8. doi: 10.1073/pnas.1305560110. Epub 2013 Aug 6.
Rubber oxygenase A (RoxA) is one of only two known enzymes able to catalyze the oxidative cleavage of latex for biodegradation. RoxA acts as a processive dioxygenase to yield the predominant product 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a tri-isoprene unit. Here we present a structural analysis of RoxA from Xanthomonas sp. strain 35Y at a resolution of 1.8 Å. The enzyme is a 75-kDa diheme c-type cytochrome with an unusually low degree of secondary structure. Analysis of the heme group arrangement and peptide chain topology of RoxA confirmed a distant kinship with diheme peroxidases of the CcpA family, but the proteins are functionally distinct, and the extracellular RoxA has evolved to have twice the molecular mass by successively accumulating extensions of peripheral loops. RoxA incorporates both oxygen atoms of its cosubstrate dioxygen into the rubber cleavage product ODTD, and we show that RoxA is isolated with O2 stably bound to the active site heme iron. Activation and cleavage of O2 require binding of polyisoprene, and thus the substrate needs to use hydrophobic access channels to reach the deeply buried active site of RoxA. The location and nature of these channels support a processive mechanism of latex cleavage.
橡胶加氧酶 A（RoxA）是仅有的两种能够催化乳胶生物降解的氧化断裂的酶之一。RoxA 作为一种连续的双加氧酶，生成主要产物 12-氧代-4,8-二甲基-十三烷-4,8-二烯-1-醛（ODTD），这是一个三异戊二烯单元。在这里，我们在 1.8Å 的分辨率下展示了来自黄单胞菌 35Y 的 RoxA 的结构分析。该酶是一种 75kDa 的双血红素 c 型细胞色素，具有异常低的二级结构程度。对 RoxA 的血红素基团排列和肽链拓扑结构的分析证实了与 CcpA 家族的双血红素过氧化物酶的亲缘关系较远，但这些蛋白质在功能上是不同的，并且通过连续积累外围环的扩展，细胞外 RoxA 已经进化为具有两倍的分子质量。RoxA 将其共底物双氧水中的两个氧原子都掺入橡胶裂解产物 ODTD 中，我们表明 RoxA 与活性位点血红素铁结合的 O2 稳定结合。O2 的激活和裂解需要多异戊二烯的结合，因此底物需要使用疏水进入通道到达 RoxA 深埋的活性位点。这些通道的位置和性质支持乳胶裂解的连续机制。
