UC Santa Barbara

Hydroxyacid oxoacid transhydrogenase (HOT) and succinate semialdehyde dehydrogenase (SSADH) are a pair of oxidoreductases that operate along pathways adjacent to the Szent–Györgyi–Krebs cycle and together catalyze the oxidation of γ hydroxybutyrate (GHB) to succinate. GHB was initially synthesized to study the neurotransmitter GABA but has become infamous as a date rape drug. Succinate is a well-known Krebs Cycle intermediate.HOT is an iron-dependent group III alcohol dehydrogenase (ADH) and the only member of this little-known, microbial-associated group found in animals. Despite its discovery in 1988, few studies on HOT have been published and the enzyme remains poorly characterized. HOT is proposed to oxidize GHB to succinate semialdehyde with concomitant reduction of a tightly bound NAD+ cofactor that does not exchange with the solvent NAD+ pool. The NAD+ cofactor is regenerated in the active site by reduction of an α ketoglutarate co-substrate to D 2 hydroxyglutarate (D2HG). Since D2HG has been identified as an oncometabolite involved in tumor progression, and changes in HOT expression levels have been detected in some cancers, HOT appears to be a potential drug target worthy of continued study. In published research on HOT, animal organs have been primary source of the enzyme, with cultured animal cells being secondary. However, structural (X ray) studies demand a relatively good amount of pure enzyme, while functional (enzyme kinetics) studies would benefit from the ability to mutate active site residues. Recombinant HOT containing an affinity purification tag, transformed into E. coli or stably transfected into a mammalian cell line, would ideally produce large amounts of pure and mutable HOT for structural and functional studies. This manuscript describes the heterologous expression of human HOT in E. coli, resulting in: 1) soluble and nearly pure recombinant HOT containing an N terminal maltose binding protein; and 2) soluble and partially purified recombinant HOT with an N terminal His-tag. Also described is the stable transfection of PC 12 cells (rat pheochromocytoma) with a vector encoding recombinant HOT with a C terminal HA tag. Integration of this sequence into the cell’s genome resulted in a line of cells that constitutively expressed this construct and, upon lysis and chromatography, yielded moderately pure recombinant HOT. Lastly, because the HOT catalyzed reaction produces no measurable change in absorbance or fluorescence under steady state conditions, the isolated reaction cannot be conveniently followed by UV-Vis or fluorescence spectroscopy. However, the HOT reaction may be coupled to the SSADH reaction since the succinate semialdehyde produced by HOT is the substrate for SSADH and the SSADH reaction produces NAD(P)H. Because NAD(P)H has a maximal absorbance at 340 nm, or fluorescence emission at 460 nm, the coupled reaction can be conveniently followed using a UV Vis spectrophotometer or a spectrofluorometer. E. coli SSADH was chosen as the auxiliary enzyme for the coupled assay, though the enzyme kinetics of this SSADH had yet to be thoroughly investigated. Thus, this dissertation also describes the expression, purification and steady-state enzyme kinetics for recombinant E. coli SSADH containing an N terminal His tag. This effort resulted in the determination of kinetic constants and pH-rate profile for the SSADH-catalyzed oxidation of succinic semialdehyde to succinate.