Crystal structure of 3-isopropylmalate dehydrogenase in complex with NAD+ and a designed inhibitor

HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB−) to grow on a minimal medium in 2 days were obtained. One of the variants LR5–1, with eight amino acid replacements, was found to possess a 65-fold increased kcat/Km value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the kcat/Km value and a partial recovery of the decreased thermotolerance of LR5–1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5–1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5–1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.

Download Full-text

Crystal Structure of 3-Isopropylmalate Dehydrogenase from the Moderate Facultative Thermophile, Bacillus coagulans: Two Strategies for Thermostabilization of Protein Structures

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a021867 ◽

1997 ◽

Vol 122 (6) ◽

pp. 1092-1104 ◽

Cited By ~ 16

Author(s):

D. Tsuchiya ◽

T. Sekiguchi ◽

A. Takenaka

Keyword(s):

Crystal Structure ◽

Protein Structures ◽

Bacillus Coagulans ◽

Facultative Thermophile ◽

Isopropylmalate Dehydrogenase

Download Full-text

Crystal Structure Determination of Glycerol-Containing Lipids from Electron Diffraction Data. Use of Analog Compounds Based upon Configurational Isomers of Cyclopentane-1, 2, 3-TRIOL

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077761 ◽

1977 ◽

Vol 35 ◽

pp. 24-25

Author(s):

Douglas L. Dorset ◽

Anthony J. Hancock

Keyword(s):

Crystal Structure ◽

Electron Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Crystal Surface ◽

Coherent Scattering ◽

Crystal Structure Determination ◽

Electron Diffraction Data ◽

Polar Group ◽

Axis Orientation

Lipids containing long polymethylene chains were among the first compounds subjected to electron diffraction structure analysis. It was only recently realized, however, that various distortions of thin lipid microcrystal plates, e.g. bends, polar group and methyl end plane disorders, etc. (1-3), restrict coherent scattering to the methylene subcell alone, particularly if undistorted molecular layers have well-defined end planes. Thus, ab initio crystal structure determination on a given single uncharacterized natural lipid using electron diffraction data can only hope to identify the subcell packing and the chain axis orientation with respect to the crystal surface. In lipids based on glycerol, for example, conformations of long chains and polar groups about the C-C bonds of this moiety still would remain unknown.One possible means of surmounting this difficulty is to investigate structural analogs of the material of interest in conjunction with the natural compound itself. Suitable analogs to the glycerol lipids are compounds based on the three configurational isomers of cyclopentane-1,2,3-triol shown in Fig. 1, in which three rotameric forms of the natural glycerol derivatives are fixed by the ring structure (4-7).

Download Full-text

Spherulitic crystal growth in the prodissoconch larval of Crassostrea virginica

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076263 ◽

1983 ◽

Vol 41 ◽

pp. 518-519

Author(s):

George G. Cocks ◽

Louis Leibovitz ◽

DoSuk D. Lee

Keyword(s):

Crystal Structure ◽

Crassostrea Virginica ◽

Crystal Orientation ◽

Developmental Changes ◽

Gastrula Stage ◽

Orthorhombic Crystal ◽

Orthorhombic Crystal Structure ◽

Stages Of Growth ◽

Early Stages ◽

Initial Deposition

Our understanding of the structure and the formation of inorganic minerals in the bivalve shells has been considerably advanced by the use of electron microscope. However, very little is known about the ultrastructure of valves in the larval stage of the oysters. The present study examines the developmental changes which occur between the time of conception to the early stages of Dissoconch in the Crassostrea virginica(Gmelin), focusing on the initial deposition of inorganic crystals by the oysters.The spawning was induced by elevating the temperature of the seawater where the adult oysters were conditioned. The eggs and sperm were collected separately, then immediately mixed for the fertilizations to occur. Fertilized animals were kept in the incubator where various stages of development were stopped and observed. The detailed analysis of the early stages of growth showed that CaCO3 crystals(aragonite), with orthorhombic crystal structure, are deposited as early as gastrula stage(Figuresla-b). The next stage in development, the prodissoconch, revealed that the crystal orientation is in the form of spherulites.

Download Full-text

Transmission Electron Microscopy studies of the vacancy ordering in YSI2-X thin films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087124 ◽

1991 ◽

Vol 49 ◽

pp. 562-563

Author(s):

F.-R. Chen ◽

T. L. Lee ◽

L. J. Chen

Keyword(s):

Crystal Structure ◽

Electron Microscopy ◽

Thin Films ◽

Diffraction Pattern ◽

Annealing Temperature ◽

Lattice Mismatch ◽

Si Substrate ◽

Metal Thin Films ◽

Transmission Electron ◽

Vacancy Ordering

YSi2-x thin films were grown by depositing the yttrium metal thin films on (111)Si substrate followed by a rapid thermal annealing (RTA) at 450 to 1100°C. The x value of the YSi2-x films ranges from 0 to 0.3. The (0001) plane of the YSi2-x films have an ideal zero lattice mismatch relative to (111)Si surface lattice. The YSi2 has the hexagonal AlB2 crystal structure. The orientation relationship with Si was determined from the diffraction pattern shown in figure 1(a) to be and . The diffraction pattern in figure 1(a) was taken from a specimen annealed at 500°C for 15 second. As the annealing temperature was increased to 600°C, superlattice diffraction spots appear at position as seen in figure 1(b) which may be due to vacancy ordering in the YSi2-x films. The ordered vacancies in YSi2-x form a mesh in Si plane suggested by a LEED experiment.

Download Full-text

Crystal Structure Analysis of Cu-Zr Alloys by Convergent Beam Diffraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098277 ◽

1981 ◽

Vol 39 ◽

pp. 354-355

Author(s):

A. F. Marshall ◽

J. W. Steeds ◽

D. Bouchet ◽

S. L. Shinde ◽

R. G. Walmsley

Keyword(s):

Crystal Structure ◽

Point Group ◽

Intermetallic Phase ◽

Three Dimensional ◽

Crystal Structure Analysis ◽

Ion Milling ◽

Composition And Structure ◽

Unit Cells ◽

Sputter Deposited ◽

Convergent Beam

Convergent beam electron diffraction is a powerful technique for determining the crystal structure of a material in TEM. In this paper we have applied it to the study of the intermetallic phases in the Cu-rich end of the Cu-Zr system. These phases are highly ordered. Their composition and structure has been previously studied by microprobe and x-ray diffraction with sometimes conflicting results.The crystalline phases were obtained by annealing amorphous sputter-deposited Cu-Zr. Specimens were thinned for TEM by ion milling and observed in a Philips EM 400. Due to the large unit cells involved, a small convergence angle of diffraction was used; however, the three-dimensional lattice and symmetry information of convergent beam microdiffraction patterns is still present. The results are as follows:1) 21 at% Zr in Cu: annealed at 500°C for 5 hours. An intermetallic phase, Cu3.6Zr (21.7% Zr), space group P6/m has been proposed near this composition (2). The major phase of our annealed material was hexagonal with a point group determined as 6/m.

Download Full-text

High resolution STEM imaging study on high Tc superconductor YBa2CuaO7-x

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100106478 ◽

1988 ◽

Vol 46 ◽

pp. 882-883

Author(s):

H.-J. Ou ◽

J. M. Cowley

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Grain Boundary ◽

Strong Field ◽

Imaging Study ◽

Structure Defects ◽

High Tc ◽

High Tc Superconductor ◽

Diffraction Patterns ◽

Lattice Planes

Using the dedicate VG-HB5 STEM microscope, the crystal structure of high Tc superconductor of YBa2Cu3O7-x has been studied via high resolution STEM (HRSTEM) imaging and nanobeam (∽3A) diffraction patterns. Figure 1(a) and 2(a) illustrate the HRSTEM image taken at 10' times magnification along [001] direction and [100] direction, respectively. In figure 1(a), a grain boundary with strong field contrast is seen between two crystal regions A and B. The grain boundary appears to be parallel to a (110) plane, although it is not possible to determine [100] and [001] axes as it is in other regions which contain twin planes [3]. Following the horizontal lattice lines, from left to right across the grain boundary, a lattice bending of ∽4° is noticed. Three extra lattice planes, indicated by arrows, were found to terminate at the grain boundary and form dislocations. It is believed that due to different chemical composition, such structure defects occur during crystal growth. No bending is observed along the vertical lattice lines.

Download Full-text

Crystal structure images of large gold clusters and growing crystals by HRTEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010011218x ◽

1984 ◽

Vol 42 ◽

pp. 428-429

Author(s):

L.R. Wallenberg ◽

J.-O. Bovin ◽

G. Schmid

Keyword(s):

Crystal Structure ◽

Nucleation And Growth ◽

Close Packing ◽

Gold Clusters ◽

Molecular Weight Determination ◽

Growth Mechanisms ◽

Starting Point ◽

Different Types ◽

Nucleation And Growth Mechanisms ◽

Points Of View

Metallic clusters are interesting from various points of view, e.g. as a mean of spreading expensive catalysts on a support, or following heterogeneous and homogeneous catalytic events. It is also possible to study nucleation and growth mechanisms for crystals with the cluster as known starting point.Gold-clusters containing 55 atoms were manufactured by reducing (C6H5)3PAuCl with B2H6 in benzene. The chemical composition was found to be Au9.2[P(C6H5)3]2Cl. Molecular-weight determination by means of an ultracentrifuge gave the formula Au55[P(C6H5)3]Cl6 A model was proposed from Mössbauer spectra by Schmid et al. with cubic close-packing of the 55 gold atoms in a cubeoctahedron as shown in Fig 1. The cluster is almost completely isolated from the surroundings by the twelve triphenylphosphane groups situated in each corner, and the chlorine atoms on the centre of the 3x3 square surfaces. This gives four groups of gold atoms, depending on the different types of surrounding.

Download Full-text