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Leucine Dehydrogenase (leucine + dehydrogenase)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

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

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002
Tatyana A. Muranova
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 VM 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 VM 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. [source]

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesis

FEBS JOURNAL, Issue 22 2001
Xing-Guo Wang
In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards l -methionine, l -norleucine and l -norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used l -aspartate and l -leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher Km values for glutamate/2-oxoglutarate at pH 7.0, but a similar kcat/Km value and lower Km at pH 8.0, and a nearly 22-fold lower S0.5 (substrate concentration giving half-saturation under conditions where Michaelis,Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100,1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially l -norleucine and l -methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate kcat and Km separately, but for reductive amination the additional mutations have no significant effect on kcat at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in Km. At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine ,,2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants. [source]

Stereoselective synthesis of L-[15N] amino acids with glucose dehydrogenase and galactose mutarotase as NADH regenerating system

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 4 2008
Maria Chiriac
Abstract We have developed an efficient stereospecific enzymatic synthesis of L-[15N]-valine, L-[15N]-leucine, L-[15N]-norvaline, L-[15N]-norleucine and L-[15N]-isoleucine from the corresponding ,-keto acids by coupling the reactions catalysed by leucine dehydrogenase and glucose dehydrogenase/galactose mutarotase. Giving high yields of L-amino acids, the procedure is economical and easy to perform and to monitor at a synthetically useful scale (1,10,g). Copyright © 2008 John Wiley & Sons, Ltd. [source]