The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa αβ heterodimer in an undissociated form
The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa αβ heterodimer in an undissociated form
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DOI：
10.1046/j.1432-1033.2003.03561.x
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发表时间：
2003-05-01
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影响因子：
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通讯作者：
Martín, JF
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作者：
Fernández, FJ;Cardoza, RE;Martín, JF
构巢曲霉和产黄青霉的异青霉素 N 酰基转移酶 (IAT) 在以未解离形式维持 40-kDa 酰基转移酶原异二聚体的能力方面存在差异。通过凝胶过滤，天然构巢曲霉 IAT 显示出 40 kDa 的分子量。通过凝胶过滤显示 P. chrysogenum IAT 的分子量为 29 kDa（对应于酶的 β 亚基），但即使在粗提物中也从未观察到未解离的 40-kDa 异二聚体。异源表达实验表明，IAT的色谱行为是由表达实验中使用的pen DE基因的来源决定的，而不是由宿主本身决定的。当构巢曲霉的pen DE基因在P.chrysogenum npe 6和npe 8或Acremium chrysogenum中表达时，形成的IAT的分子量为40kDa。另一方面，当源自P.chrysogenum的pen DE基因在A.chrysogenum中表达时，活性IAT的分子量为29kDa。从构巢曲霉 cDNA 文库克隆并在大肠杆菌中过表达的 pen DE 基因的无内含子形式形成具有酶活性的 40-kDa proIAT，如 IAT 抗体免疫印迹所示，该基因不会自我加工。即使用构巢曲霉粗提物处理，这种 40 kDa 的蛋白质仍然未加工。相比之下，从cDNA文库克隆的P.chrysogenum pen DE无内含子基因在大肠杆菌中表达，并且IAT被有效地自我加工成其α(29kDa)和β(11kDa)亚基。结论是，P. chrysogenum 和 A. nidulans 在自我加工各自的 proIAT 蛋白以及将 α 和 β 亚基维持为未解离的异二聚体的能力方面存在差异，可能是因为 proIAT 中影响自催化活性的氨基酸序列差异。
The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40-kDa proacyltransferase alphabeta heterodimer in an undissociated form. The native A. nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration. The P. chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the beta subunit of the enzyme) but the undissociated 40-kDa heterodimer was never observed even in crude extracts. Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the pen DE gene used in the expression experiments and not by the host itself. When the pen DE gene of A. nidulans was expressed in P. chrysogenum npe 6 and npe 8 or in Acremonium chrysogenum , the IAT formed had a molecular mass of 40 kDa. On the other hand, when the pen DE gene originating from P. chrysogenum was expressed in A. chrysogenum , the active IAT had a molecular mass of 29 kDa. The intronless form of the pen DE gene cloned from an A. nidulans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40-kDa proIAT, which was not self-processed as shown by immunoblotting with antibodies to IAT. This 40-kDa protein remained unprocessed even when treated with A. nidulans crude extract. In contrast, the P. chrysogenum pen DE intronless gene cloned from a cDNA library was expressed in E. coli , and the IAT was self-processed efficiently into its alpha (29 kDa) and beta (11 kDa) subunits. It is concluded that P. chrysogenum and A. nidulans differ in their ability to self-process their respective proIAT protein and to maintain the alpha and beta subunits as an undissociated heterodimer, probably because of the amino-acid sequence differences in the proIAT which affect the autocatalytic activity.