Using site-saturation mutagenesis to explore mechanism and substrate specificity in thiamin diphosphate-dependent enzymes.

For almost 20 years, site-saturation mutagenesis (SSM) has been used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry. Much of this work has focused on enzymes such as lipases and esterases, although the range is rapidly expanding. By contrast, using SSM to study enzyme mechanisms is much less common. Instead, site-directed mutagenesis is more generally employed, with a particular emphasis on alanine variants. In the present review, we provide examples of the growing use of SSM to study not only substrate and reaction selectivity, but also the reaction mechanism of thiamin diphosphate (ThDP)-dependent enzymes. We report that the use of SSM to examine the roles of the catalytic residues of benzoylformate decarboxylase gave rise to results that were at odds with earlier kinetic and structural studies using alanine substitutions and also questioned their conclusions. SSM was also employed to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V-conformation of the ThDP cofactor is maintained. X-ray structures showed that ThDP stayed in the V-conformation even when the replacement residues were charged or did not contact the cofactor. We also summarize the results obtained when SSM was used to evolve new substrate specificity and/or enantioselectivity in ThDP-dependent enzymes such as benzoylformate decarboxylase, transketolase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase and the E1 component of the 2-oxoglutarate dehydrogenase complex.