Phe317 Is Essential for Rubber Oxygenase RoxA Activity
Phe317 Is Essential for Rubber Oxygenase RoxA Activity
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DOI：
10.1128/aem.02385-12
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发表时间：
2012-11-01
影响因子：
4.4
通讯作者：
Jendrossek, Dieter
中科院分区：
文献类型：
--
作者：
Birke, Jakob;Hambsch, Nadja;Jendrossek, Dieter
RoxA是黄单胞菌35 Y菌株在橡胶上生长期间分泌的胞外C型二血红素细胞色素。RoxA将聚（顺式-1，4-异戊二烯）裂解为12-氧代-4，8-二甲基十三碳-4，8-二烯-1-醛（ODTD）。RoxA结构的分析表明，Phe 317位于接近（约5埃）的N-末端血红素，推测代表的活性位点。为了找到Phe 317是否对催化作用重要的证据，我们将其改为酪氨酸、色氨酸、亮氨酸、组氨酸或丙氨酸。所有五种RoxA突变蛋白均在各自的基因整合到黄单胞菌Delta roxA菌株的染色体中后表达。对于表达Phe 317 Leu、Phe 317 Ala或Phe 317 His变体（野生型> Leu > Ala > His）的黄单胞菌属菌株，发现在不透明乳胶琼脂上形成残留的透明带。Phe 317变为酪氨酸或色氨酸的菌株无活性。纯化Phe 317 Ala和Phe 312 Leu RoxA突变蛋白，聚异戊二烯裂解活性分别降低至约3%和10%。RoxA突变蛋白的紫外-可见光谱证实，这两个血红素基团都存在于氧化形式，但光谱响应添加低分子量（抑制）配体分子，如咪唑和吡啶是不同的野生型RoxA。我们的研究结果表明，残基317参与与底物的相互作用。这是第一次报告的结构-功能分析的聚异戊二烯裂解酶和鉴定的氨基酸是必不可少的聚异戊二烯裂解活性。
RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (approximate to 5 angstrom) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. Delta roxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to approximate to 3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.