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Probing the Role of Highly Conserved Residues Forming the Acceptor Binding Pocket of the Promiscuous Glycosyltransferase MGT in Defining the Specificity towards a Panel of Flavonoids

Chenying Xie1#, Weiqing Han2#, Peng George Wang1, and Jiansong Cheng1*

1College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research and State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, PR China; E-mail: jiansongcheng@nankai.edu.cn

2State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China

# These authors contributed equally to this work.

* To whom correspondence should be addressed.

Received December 6, 2012; Revision received January 27, 2013
MGT, a macrolide UDP-glycosyltransferase from Streptomyces lividans, has been employed as a synthetic “tool kit” to synthesize a series of modified antibiotics owing to its broad substrate plasticity. Other than MGT, a number of UDP-glycosyltransferases with substrate promiscuity were also used to alter glycosylation patterns of secondary metabolites in an emerging method called “chemoenzymatic glycorandomization”. However, the structural basis of tolerating variant acceptors for these glycosyltransferases is still not clear. In this study, the relationship between the amino acid residues forming the acceptor binding pocket and the specificity of an MGT was investigated in evolutionary and structural aspects. Interestingly, alterations of the volume and hydrophobic environment of the binding pocket by replacing Ile127 or Val151 with a bulky Phe conferred on the MGT novel activities for glycosylating flavonoids that are not accepted by the wild-type MGT.
KEY WORDS: glycosyltransferase, specificity, mutagenesis, macrolide, flavonoid

DOI: 10.1134/S000629791305012X