Expression, Purification, and Characterization of Two N,N-Dimethyltransferases, TylM1 and DesVI, Involved in the Biosynthesis of Mycaminose and Desosamine
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- 作者：Huawei Chen ; Hiroshi Yamase ; Kazuo Murakami ; Cheng-wei Chang ; Lishan Zhao ; Zongbao Zhao ; and Hung-wen Liu
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 23, 2002
- 年：2002
- 卷：41
- 期：29
- 页码：9165 - 9183
- 全文大小：238K
- 年卷期：v.41,no.29(July 23, 2002)
- ISSN：1520-4995
文摘
Methylation catalyzed by an S-adenosylmethionine- (AdoMet-) dependent methyltransferaseis an effective means to alter the hydrophilicity and/or nucleophilicity of a molecule. While a large numberof 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 foundin several macrolide antibiotics, such as tylosin and methymycin, respectively. Both sugars contain a C-3N,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 tylosinbiosynthetic gene cluster and desVI in the methymycin biosynthetic gene cluster as genes encoding thecorresponding N,N-dimethyltransferases. To verify the proposed roles of the tylM1 and desVI genes, wehave overexpressed and purified their encoded products, synthesized the predicted substrates, andcharacterized the catalytic function of these proteins. Our studies showed that TylM1 and DesVI arehomodimeric proteins and have nearly identical biochemical properties. These enzymes do not have strongpreference for binding either the unmethylated substrate or the monomethylated intermediate. It is thechemical reactivity of the nitrogen functional group that determines the relative rate of a particularmethylation step. Thus, our results not only establish TylM1 and DesVI as new members of a smallfamily of enzymes that are capable of catalyzing N,N-dimethylation of an amino group but also provideevidence indicating that the methylation catalyzed by AdoMet-dependent methyltransferases proceeds ina stepwise manner and is nucleophilic in nature.