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首页> 外文期刊>BMC Microbiology >Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum
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Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

机译：产黄青霉青霉菌penDE基因的真菌基因旁系同源物的分子表征

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Background Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway. Results The IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null ial mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic) and amidohydrolase (6-APA forming) activities in vivo. When the canonical ARL motif (leading to peroxisomal targeting) was added to the C-terminus of the IAL protein (IALARL) by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in Escherichia coli. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive in vitro. Conclusion No activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the P. chrysogenum IAL, the A. nidulans IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the ial gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in A. nidulans, the ial and penDE genes might have been formed from a single ancestral gene that became duplicated during evolution, although a separate evolutive origin for the ial and penDE genes, is also discussed.
机译：背景产黄青霉通过过氧化物酶体IPN酰基转移酶（IAT）将异青霉素N（IPN）转化为疏水性青霉素，该酶由penDE基因编码。在对产黄青霉基因组的计算机分析中，发现存在基因Pc13g09140，该基因最初被描述为编码IAT的penDE基因的旁系同源物。我们称此基因为ial，因为它编码的蛋白与IAT高度相似（IAL类似于IAT）。我们进行了一项调查，以表征ial基因并确定IAL蛋白在青霉素生物合成途径中的作用。结果IAL包含IAT的特征基序，例如加工位点，但缺少过氧化物酶体靶向序列ARL。无效的ial突变体和过表达的菌株表明，IAL在体内缺乏酰基转移酶（青霉素的生物合成）和酰胺水解酶（6-APA形成）活性。当通过定点诱变将典型的ARL基序（导致过氧化物酶体靶向）添加到IAL蛋白（IAL ARL ）的C端时，未检测到青霉素的生物合成活性。由于IAT仅在将前蛋白精确自我加工成α和β亚基后才有活性，因此在大肠杆菌中测试了IAL的自我加工。过表达实验和SDS-PAGE分析表明，IAL也是两个亚基自我加工的，但是尽管加工正确，但该酶在体外仍然没有活性。结论IAL中未检测到与青霉素生物合成相关的活性。产黄青霉IAL，构巢曲霉IAL同源物和IAT之间的序列比较表明，酶活性的缺乏似乎是由于硫酯酶活性位点中必需的Ser309的改变。在包括非青霉素生产者在内的许多其他子囊菌中也发现了ial基因的同源物。我们的数据表明，像在构巢曲霉中一样，ial和penDE基因可能是由单个祖先基因形成的，而该祖先基因在进化过程中被复制，尽管也讨论了ial和penDE基因的独立进化起源。

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