Sector of Organic Chemistry and Biochemistry, University of Ioannina, Ioannina, Greece.
Appl Environ Microbiol. 2012 Feb;78(3):621-7. doi: 10.1128/AEM.07137-11. Epub 2011 Nov 18.
A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent K(m) of 35 μM for Diox1 and 29 μM for Diox2, whereas they showed similar kinetic turnover characteristics with K(cat)/K(m) values of 11 × 10(6) M(-1) s(-1) and 12 × 10(6) M(-1) s(-1), respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans.
从分离自希腊杂酚油污染地点的 Arthrobacter phenanthrenivorans sp. nov. 菌株 Sphe3 中，通过离子交换、疏水性相互作用和凝胶过滤层析，纯化出一种具有 1-羟基-2-萘甲酸（1-H2NA）双加氧酶活性的蛋白质级分。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和串联质谱（MS-MS）分析表明，通过 SDS-PAGE 和银染分析的主要 45 kDa 蛋白质物种的寡肽氨基酸序列，占整个序列的 29%，与 Nocardioides sp. 株 KP7 的 1-H2NA 双加氧酶表现出强烈的同源性。对最近测序的 Sphe3 基因组的 BLAST 搜索显示了两个假定的开放阅读框，分别命名为 diox1 和 diox2，彼此之间的核苷酸同一性为 90%，与 Nocardia sp. 同源物的氨基酸同一性为 85%。diox1 位于 Sphe3 天然质粒上，而 diox2 位于染色体上。这两个基因都被作为唯一碳源和能源的菲诱导，并且与预期的一样，都受到碳分解代谢物阻遏的影响。染色体（diox2）基因的相对 RNA 转录水平明显高于其质粒（diox1）同源物。扩增、克隆并在大肠杆菌中过表达了两个 diox1 和 diox2 假定基因。重组大肠杆菌细胞表达了 1-H2NA 双加氧酶活性。重组酶表现出 Michaelis-Menten 动力学，Diox1 的表观 K(m)为 35 μM，Diox2 的表观 K(m)为 29 μM，而它们的动力学转换特征相似，K(cat)/K(m) 值分别为 11×10(6) M(-1) s(-1)和 12×10(6) M(-1) s(-1)。Sphe3 基因组中存在两个 diox1 和 diox2 同源物表明，复制转位事件导致了 A. phenanthrenivorans 中 1-H2NA 双加氧酶的进化。
