Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum.
Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum.
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DOI：
10.1186/1471-2180-9-104
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发表时间：
2009-05-26
期刊：
影响因子：
4.2
通讯作者：
Martín JF
中科院分区：
文献类型：
--
作者：
García-Estrada C;Vaca I;Ullán RV;van den Berg MA;Bovenberg RA;Martín JF
产黄青霉通过由penDE基因编码的过氧化物酶体IPN酰基转移酶（IAT）将异青霉素N（IPN）转化为疏水性青霉素。对P. chrysogenum基因组的计算机分析揭示了一个基因Pc 13 g 09140的存在，该基因最初被描述为编码IAT的pendE基因的paramino。我们称这个基因为IAL，因为它编码一种与IAT高度相似的蛋白质（IAL为IAT样）。我们已经进行了一项调查，以表征ial基因，并确定在青霉素生物合成途径的IAL蛋白的作用。IAL含有IAT的特征性基序，例如加工位点，但缺乏过氧化物酶体靶向序列ARL。突变体和过表达菌株表明IAL在体内缺乏酰基转移酶（青霉素生物合成）和酰胺水解酶（6-阿帕形成）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（IALARL）的C-末端时，未检测到青霉素生物合成活性。由于IAT仅在前蛋白精确自加工成α和β亚基后才有活性，因此在大肠杆菌中测试了IAL的自加工。过表达实验和SDS-PAGE分析表明，IAL也是自我加工的两个亚基，但尽管正确的加工，酶在体外保持无活性。未检测到IAL的青霉素生物合成相关活性。对产黄青霉IAL、A. nidulans IAL同源物和IAT，揭示了酶活性的缺乏似乎是由于硫酯酶活性位点中必需的Ser 309的改变。ial基因的同源物已经在许多其他子囊菌中发现，包括非青霉素生产者。我们的数据表明，像在A。虽然ial和penDE基因是独立的进化起源，但ial和penDE基因可能是由一个在进化过程中复制的祖先基因形成的。
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. 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. 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.
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影响因子：
4.1
作者：
APLIN, RT;BALDWIN, JE;SCHOFIELD, CJ
通讯作者：
SCHOFIELD, CJ
DOI：
10.1046/j.1432-1033.2003.03561.x
发表时间：
2003-05-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Fernández, FJ;Cardoza, RE;Martín, JF
通讯作者：
Martín, JF
影响因子：
4.4
作者：
Laich, F;Fierro, F;Martín, JF
通讯作者：
Martín, JF
影响因子：
4.1
作者：
Lamas-Maceiras, M;Vaca, I;Martín, JF
通讯作者：
Martín, JF
影响因子：
4.9
作者：
ALVAREZ, E;CANTORAL, JM;MARTIN, JF
通讯作者：
MARTIN, JF