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首页> 外文期刊>Journal of bacteriology >Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum.
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Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum.

机译：产黄青霉异青霉素N-酰基转移酶基因（penDE）中三个功能丧失突变的分子特征。

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Five mutants of Penicillium chrysogenum blocked in penicillin biosynthesis (npe) which are deficient in isopenicillin N-acyltransferase were isolated previously. Three of these mutants, npe6, npe7, and npe8, have been characterized at the molecular level and compared with npe10, a deletion mutant. Transcripts of normal size (1.15 kb) of the penDE genes, which encode isopenicillin N-acyltransferase, and also of the pcbAB (11.5 kb) and pcbC (1.1 kb) genes were observed in all mutants except for the npe10 mutant. Immunoblotting studies using antibodies against isopenicillin N-acyltransferase showed that all mutants (except npe10) formed the 40-kDa (unprocessed) protein and the 29-kDa subunit of the isopenicillin N-acyltransferase. The 11-kDa subunit could not be observed in the immunoblots. The mutant penDE genes of strains npe6, npe7, and npe8 were cloned and sequenced. These three strains showed a mutation in the penDE genes which results in a single amino acid change in each modified isopenicillin N-acyltransferase. The mutation in npe6 resulted in a change of Gly-150 to Val, whereas the mutation in both npe7 and npe8 introduced a change of Glu-258 to Lys. Replacement of the Val-150 and Lys-258 mutations by constructing hybrid isopenicillin N-acyltransferase molecules led to the recovery of the isopenicillin N-acyltransferase activity. The mutations in npe6, npe7, and npe8 do not affect the ability of the 40-kDa isopenicillin N-acyltransferase to be processed into the component subunits.
机译：先前已分离出五个在青霉素生物合成（npe）中受阻的产黄青霉突变体，它们缺少异青霉素N-酰基转移酶。这些突变体中的三个，即npe6，npe7和npe8，已在分子水平上进行了表征，并与缺失突变体npe10进行了比较。除npe10突变体外，在所有突变体中均观察到了编码异青霉素N-酰基转移酶的penDE基因的正常大小（1.15 kb）的转录本，以及pcbAB（11.5 kb）和pcbC（1.1 kb）的转录本。使用针对异青霉素N-酰基转移酶的抗体进行的免疫印迹研究表明，所有突变体（npe10除外）均形成了异青霉素N-酰基转移酶的40 kDa（未加工）蛋白和29 kDa亚基。在免疫印迹中未观察到11 kDa亚基。克隆并测序了菌株npe6，npe7和npe8的突变penDE基因。这三个菌株在penDE基因中显示出突变，其导致每个修饰的异青霉素N-酰基转移酶中的单个氨基酸变化。 npe6中的突变导致Gly-150变为Val，而npe7和npe8中的突变均导致Glu-258变为Lys。通过构建杂种异青霉素N-酰基转移酶分子替换Val-150和Lys-258突变导致异青霉素N-酰基转移酶活性的恢复。 npe6，npe7和npe8中的突变不影响40 kDa异青霉素N-酰基转移酶被加工成组分亚基的能力。

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