From the Department of Pharmacology and the Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232.
J Biol Chem. 2014 May 9;289(19):13101-11. doi: 10.1074/jbc.M114.555904. Epub 2014 Mar 21.
In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as "cyclooxygenase-2," appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.
在探索过氧化氢酶相关血红素蛋白对脂肪酸过氧化物反应的范围的过程中,我们在蓝细菌集胞藻 PCC 73102 中发现了一个新的候选物。紧邻的上游基因,注释为“环氧化酶-2”,似乎是一种潜在的脂肪酸血红素加氧酶。我们克隆了这两个基因,并在大肠杆菌中表达了 cDNA,证实了它们的血红素蛋白特性。氧电极记录表明,血红素加氧酶与油酸和亚油酸的反应非常迅速(>100 转/秒)。HPLC,包括手性柱分析、氧代产物的 UV 和 GC-MS,鉴定出一种新型的 10S-加氧酶活性。过氧化氢酶相关的血红素蛋白与亚油酸 10S-过氧化物(>2500 转/秒)快速且特异性地反应,具有针对 10S-过氧物手性对映体的过氧化物裂合酶活性。产物通过 NMR 鉴定为(8E)10-氧代-癸烯酸和 C8 片段 1-辛烯-3-醇和 2Z-辛烯-1-醇,比例约为 3:1。手性 HPLC 分析确立了在形成 3R 醇构型(99%对映体过量)方面的严格酶控制,这与亚油酸 10S-过氧化物与血红素或亚铁离子反应形成的外消旋 1-辛烯-3-醇形成对比。集胞藻亚油酸 10S-加氧酶的序列包含环氧化酶和真菌亚油酸加氧酶的特征催化序列(YRWH),似乎是血红素加氧酶的祖先。裂合酶的新活性扩展了过氧化氢酶相关蛋白的已知反应,并在 Nostoc 中特异性转化 10S-过氧亚油酸的功能。