Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermedius

Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD⁺. Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an `open' and `closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD⁺ have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-­ketoisocaproate belong to space group P2₁2₁2₁, with approximate unit-cell parameters a = 106, b = 118, c = 320 Å and an octamer in the asymmetric unit, corresponding to a V_M of 3.1 Å³ Da^-1. The crystals of the non-productive ternary complex belong to space group P6₁ or P6₅, with approximate unit-cell parameters a = b = 117, c = 502 Å and an octamer in the asymmetric unit, corresponding to a V_M of 3.0 Å³ Da^-1. These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3 Å resolution, respectively, and are suitable for a full structure determination.