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 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.
Muranova
Tatyana A
TA
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, England.
Ruzheinikov
Sergey N
SN
Sedelnikova
Svetlana E
SE
Baker
Patrick J
PJ
Pasquo
Alessandra
A
Galkin
Andrey
A
Esaki
Nobuyoshi
N
Ohshima
Toshihisa
T
Soda
Kenji
K
Rice
David W
DW
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
D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase