Experimental and kinetic studies of the escherichia coli glucuronylsynthase : an engineered enzyme for the synthesis of glucuronides

Abstract

The E. coli glucuronylsynthase is a glycosynthase enzyme derived from E. coli {u03B2}-glucuronidase. It catalyses the conjugation of a glucopyranuronic acid unit to an alcohol acceptor. These glucuronide conjugates are important markers for doping in sports drug testing and drug metabolism in pharmaceutical trials. Up till now, the optimal glucuronylsynthase reaction conditions were unknown which may have reduced the previously reported yields. This thesis covers the development and optimisation of the glucuronylsynthase-mediated glucuronylation reaction. This was achieved by determining the enzyme kinetics associated with the glucuronylsynthase enzyme and devising a synthetic strategy to overcome the water solubility issues associated with steroidal substrates. HPLC-UV was used to monitor and quantitate the components of the glucuronylsynthase reaction required for enzyme kinetic analysis. This HPLC assay was used to determine the kinetic parameters (Km, kcat) of the {u03B1}-D-glucuronyl fluoride 51 donor and a range of acceptor substrates. Substrate inhibition and product inhibition were observed during these kinetic investigations and inhibition constants are determined in each case. The optimised glucuronylsynthase-mediated reaction conditions were also determined using HPLC-UV. Different variations (pH, temperature, enzyme concentration, substrate concentration/equivalents) were altered and their effect on enzyme activity and product yield determined by HPLC-UV. Improved reaction rates and yield were observed in the glucuronylsynthase reactions involving phenol 52 and DHEA O-(carboxymethyl)oxime 116 when applying optimised conditions. To overcome the poor water solubility of steroidal substrates, carboxymethoxylamine 115 was condensed to the ketone of DHEA 55 and testosterone 7 to synthesise ionisable oxime-analogues with greatly enhanced water solubility. This provided greater yields of the steroid glucuronide conjugates compared to the direct glucuronylsynthesis of the parent steroids. Greater enzyme efficiency was also achieved due to more concentrated reaction volumes being used. The oxime moiety was cleaved under mild titanium(III) hydrolysis to complete the synthesis of the steroid glucuronide conjugate in high overall yields in three steps. Application of this steroid solubilisation strategy on a half gram scale was demonstrated. A solid-phase extraction procedure was adapted to a small library of steroids to isolate a variety of purified steroid glucuronides. This screen illustrated the broad application of the enzyme and served as a trial for a larger screen of substrates. The combination of the optimised methodology and screening procedures sets the foundation to expand the substrate repertoire for the glucuronylsynthase reaction. Upon reviewing the glucuronylsynthase procedure and alternate means of glucuronylation, the glucuronylsynthase procedure compares well, and is better than these alternative procedures in many ways. This includes an experimental procedure that is easy to set-up, mild and applicable to a broad range of substrates. Other attributes include a short and simple donor synthesis and the absence of by-products (to date) which allows high isolated yields and purity. Show more