Mammalian 3-hydroxyisobutyrate dehydrogenase

This chapter presents methods for purification of the native rabbit liver enzyme, cloning of rat and human complementary DNAs, the construction of prokaryotic expression vectors to produce rat 3-hydroxyisobutyrate dehydrogenase (HIBADH) in Escherichia coli as a glutathione transferase-tagged protein or a six histidine-tagged protein, and the expression of recombinant rat enzyme in E. coli. HIBADH includes 6-phosphogluconate dehydrogenase, D-phenylserine dehydrogenase, D-threonine dehydrogenase, and a number of hypothetical 3-hydroxyacid dehydrogenases encoded by unidentified microbial open reading frames. Rat liver HIBADH and Pseudomonas HIBADH have been structurally characterized at the level of complementary DNA sequences and expressed sequence tags corresponding to mouse, human, and microbial HIBADHs appear in various genetic databases. Structural and mechanistic studies of HIBADH require a ready source of recombinant enzyme that can be easily purified and manipulated by site-directed mutagenesis. Purified HIBADH is also a useful reagent for a specific assay for quantitation of 3-hydroxyisobutyrate in tissues.