Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095.
Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706.
J Biol Chem. 2013 May 17;288(20):14032-14045. doi: 10.1074/jbc.M113.465765. Epub 2013 Mar 26.
The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-β-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-(3)H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA.
曲霉菌属中的丝状真菌是机会性植物和动物病原体，能够通过产生各种次级代谢产物（包括洛伐他汀、青霉素和黄曲霉毒素）来适应环境。这些小分子的合成依赖于基因簇，这些基因簇受 LaeA 蛋白的全局调控。迄今为止，在所有研究过的致病性真菌中，LaeA 的 null 突变体显示出毒力降低，同时次级代谢产物减少。尽管 LaeA 的氨基酸序列包含 7-β-链甲基转移酶特征的基序，但尚未鉴定出 LaeA 的甲基受体底物。在这项工作中，我们使用各种测定方法（包括体内 S-腺苷甲硫氨酸-[甲基-（3）H]甲硫氨酸标记、使用假定蛋白底物的靶向体外甲基化实验，或使用在不同条件下生长的全细胞提取物的体外甲基化测定），均未在构巢曲霉中发现 LaeA 的甲基受体底物。然而，在每个实验中都表明 LaeA 自身发生甲基化。放射性标记的 LaeA 的氨基酸水解，然后进行阳离子交换和反相色谱分析，鉴定出蛋氨酸为修饰残基。定点突变表明，LaeA 的主要修饰位点是蛋氨酸 207。然而，体内互补表明，LaeA 的生物学功能不依赖于蛋氨酸 207。LaeA 是第一个在蛋氨酸残基上表现出自甲基化的蛋白。这些发现不仅表明 LaeA 可能与 S-腺苷甲硫氨酸发生新颖的化学作用，而且为 LaeA 的生理功能提供了新的见解。
