Junior Research Group for Microbial Biotechnology, Faculty of Biology and Biotechnology, Ruhr-University Bochum, 44780 Bochum, Germany.
Junior Research Group for Microbial Biotechnology, Faculty of Biology and Biotechnology, Ruhr-University Bochum, 44780 Bochum, Germany; Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
J Biotechnol. 2018 Oct 20;284:68-74. doi: 10.1016/j.jbiotec.2018.07.038. Epub 2018 Aug 4.
Optically pure hydroxy amino acids show several bioactivities and are valuable building blocks for the pharmaceutical industry. Fe(II)/α-ketoglutarate dependent dioxygenases catalyze the hydroxylation or sulfoxidation of l-amino acids with high regio- and stereoselectivity. While several β- and γ-specific enzymes have been described, only one δ-specific hydroxylase has been reported so far. Based on its similarity to the known l-leucine 5-hydroxylase from Nostoc punctiforme, an open reading frame from the cyanobacterium Anabaena variabilis was identified as putative l-leucine dioxygenase (AvLDO). Here we report the cloning and characterization of this dioxygenase. The enzyme showed a high preference for acidic conditions and moderate reaction temperatures. AvLDO catalyzed the regio- and stereoselective hydroxylation of several aliphatic amino acids in δ-position. In case of the sulfoxidation of l-methionine, AvLDO produced the opposite diastereomer than isoleucine dioxygenase. AvLDO is thus an interesting addition to the toolbox of Fe(II)/α-ketoglutarate dependent dioxygenases. On the genomic DNA of Anabaena variabilis ATCC 29413, the avldo gene is located on a gene cluster involved (2S,4S)-4-methylproline biosynthesis, which is contained in bioactive peptides often found from cyanobacteria. This fact suggests the metabolic functional role of this amino acid dioxygenase in cyanobacteria.
光学纯的羟基氨基酸具有多种生物活性，是制药工业有价值的构建块。Fe(II)/α-酮戊二酸依赖性双加氧酶以高区域和立体选择性催化 l-氨基酸的羟化或氧化脱硫。虽然已经描述了几种β-和γ-特异性酶，但迄今为止仅报道了一种δ-特异性羟化酶。基于其与已知来自 Nostoc punctiforme 的 l-亮氨酸 5-羟化酶的相似性，从蓝藻 Anabaena variabilis 中鉴定出一个开放阅读框作为假定的 l-亮氨酸双加氧酶 (AvLDO)。在这里，我们报告了该双加氧酶的克隆和特性。该酶对酸性条件和中等反应温度具有高偏好性。AvLDO 催化几种脂肪族氨基酸在 δ-位的区域和立体选择性羟化。在 l-蛋氨酸氧化脱硫的情况下，AvLDO 产生与异亮氨酸双加氧酶相反的非对映异构体。因此，AvLDO 是 Fe(II)/α-酮戊二酸依赖性双加氧酶工具包的有趣补充。在 Anabaena variabilis ATCC 29413 的基因组 DNA 上，avldo 基因位于涉及 (2S,4S)-4-甲基脯氨酸生物合成的基因簇上，该基因簇包含经常在蓝藻中发现的生物活性肽。这一事实表明了这种氨基酸双加氧酶在蓝藻中的代谢功能作用。
