Chen Huawei, Yamase Hiroshi, Murakami Kazuo, Chang Cheng-Wei, Zhao Lishan, Zhao Zongbao, Liu Hung-Wen
Division of Medicinal Chemistry, College of Pharmacy, Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Biochemistry. 2002 Jul 23;41(29):9165-83. doi: 10.1021/bi020245j.
Methylation catalyzed by an S-adenosylmethionine- (AdoMet-) dependent methyltransferase is an effective means to alter the hydrophilicity and/or nucleophilicity of a molecule. While a large number of enzymes capable of catalyzing methylation at carbon, oxygen, sulfur, and nitrogen atoms are known, only a few are able to catalyze N,N-dimethylation. Mycaminose and desosamine are aminohexoses found in several macrolide antibiotics, such as tylosin and methymycin, respectively. Both sugars contain a C-3 N,N-dimethylamino group which has been shown to confer the biological activity of these unusual sugars. Recently, sequence analysis as well as genetic studies has led to the assignment of tylM1 in the tylosin biosynthetic gene cluster and desVI in the methymycin biosynthetic gene cluster as genes encoding the corresponding N,N-dimethyltransferases. To verify the proposed roles of the tylM1 and desVI genes, we have overexpressed and purified their encoded products, synthesized the predicted substrates, and characterized the catalytic function of these proteins. Our studies showed that TylM1 and DesVI are homodimeric proteins and have nearly identical biochemical properties. These enzymes do not have strong preference for binding either the unmethylated substrate or the monomethylated intermediate. It is the chemical reactivity of the nitrogen functional group that determines the relative rate of a particular methylation step. Thus, our results not only establish TylM1 and DesVI as new members of a small family of enzymes that are capable of catalyzing N,N-dimethylation of an amino group but also provide evidence indicating that the methylation catalyzed by AdoMet-dependent methyltransferases proceeds in a stepwise manner and is nucleophilic in nature.
由S-腺苷甲硫氨酸（AdoMet）依赖性甲基转移酶催化的甲基化是改变分子亲水性和/或亲核性的有效手段。虽然已知大量能够催化碳、氧、硫和氮原子甲基化的酶，但只有少数能够催化N，N-二甲基化。霉胺糖和去氧胺糖分别是在几种大环内酯类抗生素（如泰乐菌素和酒霉素）中发现的氨基己糖。这两种糖都含有一个C-3 N，N-二甲基氨基，已证明该基团赋予这些特殊糖类生物活性。最近，序列分析以及遗传学研究已将泰乐菌素生物合成基因簇中的tylM1和酒霉素生物合成基因簇中的desVI指定为编码相应N，N-二甲基转移酶的基因。为了验证tylM1和desVI基因的推测功能，我们对它们编码的产物进行了过表达和纯化，合成了预测的底物，并对这些蛋白质的催化功能进行了表征。我们的研究表明，TylM1和DesVI是同二聚体蛋白，具有几乎相同的生化特性。这些酶对未甲基化的底物或单甲基化的中间体的结合没有强烈偏好。是氮官能团的化学反应性决定了特定甲基化步骤的相对速率。因此，我们的结果不仅将TylM1和DesVI确立为能够催化氨基N，N-二甲基化的小酶家族的新成员，还提供了证据表明由AdoMet依赖性甲基转移酶催化的甲基化以逐步方式进行且本质上是亲核的。
