Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47
Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47
复制标题
DOI：
10.1128/jb.00874-09
复制
发表时间：
2010-01-01
影响因子：
3.2
通讯作者：
Meckenstock, Rainer U.
中科院分区：
文献类型：
--
作者：
Selesi, Drazenka;Jehmlich, Nico;Meckenstock, Rainer U.
高度富集的δ-变形菌培养物N47厌氧氧化多环芳烃萘和2-甲基萘，硫酸盐作为电子受体。结合基因组测序和液相色谱-串联质谱法为基础的鸟枪蛋白质组分析进行，以确定基因和蛋白质参与厌氧芳香族catenase。对2-甲基萘生长的N47细胞进行蛋白质组分析，鉴定出了催化2-甲基萘无氧转化为2-萘甲酰基辅酶A（2-萘甲酰基-CoA）以及2-萘甲酰基-CoA还原性环切割的推定酶，从而导致乙酰基-CoA和CO2的形成。甘氨酰自由基催化的富马酸加成至2-甲基萘的甲基由萘基-2-甲基琥珀酸合酶（Nms）催化，所述Nms由基因nmsABC编码的α-、β-和γ-亚基组成。位于nmsABC上游的是nmsD，编码Nms活化酶，其具有S-腺苷甲硫氨酸自由基酶的特征性[Fe 4S 4]簇序列基序。bns基因簇编码参与β-氧化反应的酶，将萘基-2-甲基-琥珀酸转化为2-萘甲酰基-CoA，在下游发现4个插入开放阅读框。该簇由对应于8.1 kb的8个基因（bnsABCDEFGH）组成，其与在发酵剂“芳香族芳香族”EbN 1、Azoarcus sp.菌株T和Thauera aromatica内参与厌氧甲苯降解的酶的基因密切相关。另一个连续的DNA序列含有2-萘酰辅酶A还原酶（ncr）基因和16个在2-甲基萘生长的细胞中表达的其他基因。这些基因编码的酶被认为是催化脱芳构化和环裂解反应，将2-萘酰辅酶A转化为乙酰辅酶A和CO2。四个编码亚基（ncrABCD）的比较序列分析表明，该基因产物具有最接近的相似性Azoarcus型的苯甲酰辅酶A还原酶。目前的工作提供了第一个深入了解厌氧2-甲基萘代谢的遗传基础，并提供了解污染物降解的影响。
The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO2. The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of alpha-, beta-, and gamma-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe4S4] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers "Aromatoleum aromaticum" EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetylCoA and CO2. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.