Abstract
Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (rs/rh) from 0.33 to 1.45–2.71. Further increase in rs/rh was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5–10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.
Highlights
Glycosylation is an important source of structural diversity of natural products
To be able to utilize the vast array of Glycoside hydrolases (GH) for creation of TG, we need to understand what governs their propensity for transglycosylation or hydrolysis
We show that transglycosylation is highly dependent on pH for the wild-type β-glucosidase from Thermotoga neapolitana, it can be changed by single mutations
Summary
Glycosylation is an important source of structural diversity of natural products. It can alter the properties of compounds in a multitude of ways, e.g., changing the flavor or smell (Ribeiro 2011; Roitner et al 1984), improving water solubility (Chen et al 2011) or stability (Yamamoto et al 1990), or reducing skin irritation (Kurosu et al 2002). Glycosylation is mainly performed by Leloir glycosyltransferases. They are not well suited for glycosylation in vitro as they require expensive nucleotide-activated sugars as glycosyl donors and are often difficult to express. The use of GH for glycosylation is impaired by their naturally dominant hydrolytic activity
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