Biochemical studies of a β-1,4-rhamnoslytransferase from Streptococcus pneumonia serotype 23F

• Published in: Organic & biomolecular chemistry 2019-01, Vol.17 (5), p.1071-1075
• Main Authors: Wang, Hong, Li, Siqiang, Xiong, Chenghe, Jin, Guoxia, Chen, Zonggang, Gu, Guofeng, Guo, Zhongwu
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - metabolism; Binding sites; Chemical synthesis; Cloning; Disaccharides; Enzymatic activity; Enzymes; Hexosyltransferases - metabolism; Kinetics; Magnetic Resonance Spectroscopy - methods; Metal ions; NMR; Nuclear magnetic resonance; Oligosaccharides; Optimization; Organic chemistry; Pneumonia; Primers; Proteins; Reaction kinetics; Rhamnose - metabolism; Site-directed mutagenesis; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Streptococcus pneumoniae; Streptococcus pneumoniae - enzymology; Substrate Specificity; Substrates
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/c8ob02795a

A new β-rhamnoslytransferase Cps23FT from Streptococcus pneumonia serotype 23F was expressed and characterized. Its enzymatic activity and function were confirmed for the first time by utilizing enzymatically prepared dTDP-Rha and chemically synthesized Glcα-PP-(CH2)11-OPh as substrates. This reaction gave the desired disaccharide Rhaβ-1,4-Glcα-PP-(CH2)11-OPh in a good isolated yield (67%), suggesting the potential of Cps23FT as a tool enzyme for the synthesis of complex oligosaccharides containing difficult β-rhamnosyl linkages. Furthermore, site-directed mutagenesis of Cps23FT disclosed that its 271DKD273 motif was critical for the enzymatic activity and most likely the binding site for the required divalent metal cation.