A mutant glycosidase for oligosaccharide synthesis

S.M. Hancock, K.E. McAuley1 and B.G. Davis

Chemistry Research Laboratory, University of Oxford, Mansfield Road, OX1 3TA Oxford, UK 1Diamond Light Source, Chilton, OX11 0DE Didcot, UK

Oligosaccharides play key roles in biological recognition and signalling mechanisms and in recent years there has been a growing interest in the development of carbohydrate-based drugs.1-5 The potential therapeutic role of carbohydrate-based drugs has so far been underexploited, largely due to the complexities in their chemical synthesis. Carbohydrate-processing enzymes can be used as an alternative to chemical synthesis and have the advantage that the reactions they catalyse are regio- and stereo- specific.6

We are exploring the creation of unique glycosidase catalysts, compatible with high temperature and organic solvents, which will form glycosidic linkages between any two monosaccharides in one step. We have been investigating glycoside formation with the retaining -glycosidase from Sulfolobus solfataricus (Ss G).7, 8 Ss G is thermophilic, and displays tolerance to organic solvents. These attributes highlight the potential of this enzyme as a universal glycosylation catalyst. With the prospect of creating more versatile catalysts for the carbohydrate chemist's toolkit, we explored the redesign of the enzyme's catalytic machinery by mutagenesis, targeting enzyme mechanism.

Figure 1. Ss G, a Family 1 glycosyl hydrolase (pdb: 1gow).9 The active site nucleophile and general acid/base residues are shown in blue and red, respectively.

Family 1 glycosyl hydrolases contain two glutamates in the active site, one of which acts as the catalytic nucleophile and the other as general acid/base. Using rational mutagenesis, a mutant of Ss G has been constructed that synthesises oligosaccharides in excellent yields (UK priority patent application no. 0329011.1). We have been using a combined approach of X-ray crystallography, MS trapping experiments and kinetic studies in order to understand the mechanism by which the enzyme catalyses this process.

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OHO O

NO2

OH

OH OH

OO

OH

OHO

OH

OHOH OH

O

NO2

OH

OHO

OH

OHO

OHOHO

OH OH

O

NO2

OO

HOOH

OHO

OH

OH OH

OHO O

HO

OHO

OHO

OH OH

HOOH

O

51% 54%

25%72%

OOPh

OH

OHHOHO

OHOHO

HO OH

OPh

Figure 2. Synthesis of oligosaccharides with mutant Ss G.

The crystal structure of the mutant Ss G revealed a local rearrangement of several key residues in the active site of the protein. Two acetate ions, arising from the crystallisation conditions, were identified in the electron density in the active site region. These ions mimic the sugar substrate-WT Ss G interactions10 and further aided mechanistic interpretations.

The structural information we obtained, in combination with the results from the biochemical studies, has allowed us to propose a mechanism for Ss G mutant-catalysed glycosynthesis.

References[1] X. P. Wei, J. M. Decker, S. Y. Wang, et al., Nature 422, 307 (2003) [2] C. J. Bosques and B. Imperiali, Proc. Natl. Acad. Sci. USA 100, 7593 (2003) [3] K. M. Hoffmeister, E. C. Josefsson, N. A. Isaac, et al., Science 301, 1531 (2003) [4] B. G. Davis, J. Chem. Soc., Perkin Trans. 1, 3215 (1999) [5] R. A. Dwek, Chem. Rev. 96, 683 (1996) [6] B. G. Davis and S. M. Hancock, in Carbohydrates, edited by H. M. I. Osborn (Academic Press,

Oxford, 2003), p. 385. [7] K. Corbett, A. P. Fordham-Skelton, J. A. Gatehouse, et al., FEBS Lett. 509, 355 (2001) [8] S. M. Hancock, K. Corbett, A. P. Fordham-Skelton, et al., Chembiochem. 6, 866 (2004) [9] C. F. Aguilar, I. Sanderson, M. Moracci, et al., J. Mol. Biol. 271, 789 (1997) [10] J. Symersky, S. Li, M. Carson, et al., Proteins 51, 484 (2003).

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