MECHANISTIC AND STRUCTURAL STUDIES OF SACCHAROPINE DEHYDROGENASE FROM SACCHAROMYCES CEREVISIAE

Abstract

Lysine is an essential amino acid in mammals including humans. The α-aminoadipate (AAA) pathway for lysine biosynthesis is unique to fungal organisms and enzymes of this pathway are of interest as potential targets for anti-fungal drug design. Saccharopine dehydrogenase (SDH) [N6-(glutaryl-2)-L-lysine:NAD-oxidoreductase (L-lysine-forming) (EC 1.5.1.7)] catalyzes the reversible NAD-dependent oxidative deamination of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteines 205 and 249 is observed in structures of the apoenzyme and those with sulfate or oxalylglycine bound to the substrate-binding domain. However, in the structure with adenosine monophosphate bound in the dinucleotide-binding site, the disulfide bond is reduced.

In this dissertation, site-directed mutagenesis was used to generate the C205S mutant enzyme, which was characterized kinetically using a combination of steady state kinetics, pH-rate profiles, and isotope effects. The C205S mutant enzyme was also characterized crystallographically. Structures of the C205S apoenzyme and one with NADH bound in the dinucleotide binding site have been solved. To probe the acid-base chemical mechanism of saccharopine dehydrogenase, K77M and H96Q, K77M/C205S and H96Q/C205S, and K77M/ H96Q/C205S single, double, and triple mutants, respectively, were prepared and characterized via initial velocity studies and isotope effects.