Reconstitution and Characterization of a New Desosaminyl Transferase, EryCIII, from the Erythromycin Biosynthetic Pathway
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- 作者：Ho Young Lee ; Hak Suk Chung ; Chao Hang ; Chaitan Khosla ; Christopher T. Walsh ; Daniel Kahne ; and Suzanne Walker
- 刊名：Journal of the American Chemical Society
- 出版年：2004
- 出版时间：August 18, 2004
- 年：2004
- 卷：126
- 期：32
- 页码：9924 - 9925
- 全文大小：41K
- 年卷期：v.126,no.32(August 18, 2004)
- ISSN：1520-5126
文摘
EryCIII converts 
-mycarosyl erythronolide B into erythromycin D using TDP-D-desosamine as the glycosyl donor. We report the heterologous expression, purification, in vitro reconstitution, and preliminary characterization of EryCIII. Coexpression of EryCIII with the GroEL/ES chaperone complex was found to enhance greatly the expression of soluble EryCIII protein. The enzyme was found to be highly active with a kcat greater than 100 min-1. EryCIII was quite selective for the natural nucleotide sugar donor and macrolide acceptor substrates, unlike several other antibiotic glycosyl transferases with broad specificity such as desVII, oleG2, and UrdGT2. Within detectable limits, neither 6-deoxyerythronolide B nor 10-deoxymethynolide were found to be glycosylated by EryCIII. Furthermore, TDP-D-mycaminose, which only differs from TDP-D-desosamine at the C4 position, could not be transferred to MEB. These studies lay the groundwork for detailed structural and mechanistic analysis of an important member of the desosaminyl transferase family of enzymes.
-mycarosyl erythronolide B into erythromycin D using TDP-D-desosamine as the glycosyl donor. We report the heterologous expression, purification, in vitro reconstitution, and preliminary characterization of EryCIII. Coexpression of EryCIII with the GroEL/ES chaperone complex was found to enhance greatly the expression of soluble EryCIII protein. The enzyme was found to be highly active with a kcat greater than 100 min-1. EryCIII was quite selective for the natural nucleotide sugar donor and macrolide acceptor substrates, unlike several other antibiotic glycosyl transferases with broad specificity such as desVII, oleG2, and UrdGT2. Within detectable limits, neither 6-deoxyerythronolide B nor 10-deoxymethynolide were found to be glycosylated by EryCIII. Furthermore, TDP-D-mycaminose, which only differs from TDP-D-desosamine at the C4 position, could not be transferred to MEB. These studies lay the groundwork for detailed structural and mechanistic analysis of an important member of the desosaminyl transferase family of enzymes.