Leucine Dehydrogenase: Structure and Thermostability.

Abstract

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that hasalreadybeenapplied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.