title: Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermedius.
abstract: 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 P2(1)2(1)2(1), with approximate unit-cell parameters a = 106, b = 118, c = 320 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.1 A(3) Da(-1). The crystals of the non-productive ternary complex belong to space group P6(1) or P6(5), with approximate unit-cell parameters a = b = 117, c = 502 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.0 A(3) Da(-1). These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3 A resolution, respectively, and are suitable for a full structure determination.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, England.
authors.forename: Tatyana A
authors.initials: TA
authors.lastname: Muranova
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Ruzheinikov
authors.forename: Svetlana E
authors.initials: SE
authors.lastname: Sedelnikova
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Baker
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pasquo
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
authors.forename: Kenji
authors.initials: K
authors.lastname: Soda
authors.forename: David W
authors.initials: DW
authors.lastname: Rice
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D050803:Leucine Dehydrogenase; D008848:Micromonosporaceae / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase