Reconstitution and characterization of a new desosaminyl transferase, EryCIII, from the erythromycin biosynthetic pathway.
EryCIII converts alpha-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 alphaMEB. These studies lay the groundwork for detailed structural and mechanistic analysis of an important member of the desosaminyl transferase family of enzymes.
Lee
Ho Young
HY
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Chung
Hak Suk
HS
Hang
Chao
C
Khosla
Chaitan
C
Walsh
Christopher T
CT
Kahne
Daniel
D
Walker
Suzanne
S
D000606:Amino Sugars / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D016930:Saccharopolyspora / Q000201:enzymology
D000606:Amino Sugars; D001426:Bacterial Proteins; C038315:erythronolide B; C110125:desosamine; D004917:Erythromycin; D016695:Glycosyltransferases; C111821:EryCIII protein, Saccharopolyspora erythraea