Mamoru Sato

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.

A High-Speed Data-Collection System for Large-Unit-Cell Crystals using an Imaging Plate as a Detector

A high-speed data-collection system for large-unit-cell crystals is presented, using the Fuji Imaging Plate as an X-ray detector and a rotating-anode generator as the X-ray source. It is an automatic data-acquisition system that requires almost no manual intervention. The quality of data collected on the system is discussed. Merging R values ranged from 0.04 to 0.05. Compared with a four-circle diffractometer, data reproducibility was better, isomorphous/anomalous Patterson maps were almost identical in quality and data from a small-molecule crystal, cytidine, were of almost the same quality. Protein structures were refinable using the data measured on the system, the final crystallographic R value of the 2.2u2005A 3-isopropylmalatedehydrogenase structure being 0.185 and that of the 1.88u2005A Flammulina veltipes agglutinin structure being 0.199.

Molecular organization of a high molecular weight multi-protease complex from rat liver☆

A latent multifunctional protease with a molecular weight of 722,000 to 760,000 purified from rat liver cytosol has been reported. This paper reports on the structure and subunit composition of the enzyme. Electron microscopy showed that the enzyme was a ring-shaped particle of 160(+/- 7) A diameter and 110(+/- 10) A height with a small hole of 10 to 30 A diameter (1 A = 0.1 nm). Small-angle X-ray scattering analysis indicated that the enzyme had a prolate ellipsoidal structure with an ellipsoid cavity in the center. The maximum dimension of the enzyme was estimated to be 210 A from a pair-distance distribution function. The radius of gyration obtained from a Guinier plot and the Stokes radius based on the ellipsoidal model were 66 A and 76 A, respectively. On two-dimensional gel electrophoresis, the purified enzyme separated into 13 to 15 characteristic components with molecular weights of 22,000 to 33,000 and isoelectric points of 4 to 9. These multiple components were not artifacts produced by limited proteolysis during purification of the enzyme, because the cell-free translation products in a reticulocyte lysate with poly(A)-mRNA of rat liver consisted of multiple components of similar sizes, and because peptide mapping analyses with lysylendopeptidase and V8 protease demonstrated clear differences in the primary structures of these components. The 13 main components were isolated from the purified enzyme by reverse-phase high performance liquid chromatography and shown to be non-identical. A model of the enzyme is proposed on the basis of these observations and previous physicochemical studies. Interestingly, the morphology of this protease is similar to that of the 16 to 22 S ring-shaped particles found in a variety of eukaryotic organisms. The structural similarity between this multi-protease complex and various reported subcellular particles is discussed.

Crystallization and preliminary X-ray studies of Bacillus pumilus IPO xylanase.

The xylan-degrading enzyme xylanase, from Bacillus pumilus IPO, has been crystallized. The crystals are monoclinic, space group P21 with a = 40.8 A, b = 66.8 A, c = 34.7 A and beta = 103.0 degrees. The asymmetric unit contains one molecule of Mr 22,500. The crystals diffract to at least 2.5 A resolution, and they are suitable for X-ray crystal structure analysis at high resolution.

The α3β3 and α1β1 Complexes of ATP Synthase

ATP synthase (FoF1) is an ion-motive ATPasel that catalyzes oxidative and photosynthetic phosphorylation.1-5 The enzyme uses the energy generated by the redox chain to synthesize ATP via an electrochemical potential difference of protons (Aii;H+) across the membrane.^^ In fact, the proton current caused by FoFl generating the ApH+ was demonstrated in both liposomes6 and planar lipid bilayers.7 When the Fo portion alone is incorporated into the lipid bilayers, passive transport of H+ was demonstrated. FoF, is a multisubunit complex composed of two subcomplexes, a soluble ATPase, F1, and an ion channel, F0.*s5 Although FoFls of mitochondria, chloroplasts, and most bacterial plasma membranes are H+-motive ATPases containing H+-specific Fo, there also exists a Na+-motive ATPase containing a special F0.8 When this Na+-specific Fo is connected to the F1 of H+-motive FoFI, an Na+-motive ATPase is reconstituted? Thus, the F1 portion is an ATPase that supplies energy to the Fo portion, perhaps via a conformational change. F1 is composed of five types of subunits, a, p, y, 8, and c subunits. The nucleotide binding sites of F1 are localized on both the a and fi subunits; however, the central structure, composed of ap complexes, has not yet been isolated. This report presents the basic structures of F1, which are the a3Ps hexamer (oligomer) and alp1 heterodimer (protomer). A large conformational change was found on the a

Preliminary X‐ray crystallographic analysis of tryptophanase from Escherichia coli

complex induced by ATP-ADP binding.

Preliminary x-ray studies on Pseudomonas isoamylase.

Tryptophanase (L‐tryptophan indole‐lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P41212 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 Å, and c = 232.2 Å, with two subunits per asymmetric unit. The crystals diffract to at least 3 Å resolution, and are suitable for X‐ray structural analysis.

Crystal and molecular structure of 6-0-acetylgrayanotoxin III

Abstract Single crystals of Pseudomonas isoamylase ( M r 95,000) belong to space group P 2 1 2 1 2 1 with unit cell dimensions of a = 137.9 A , b = 52.9 A , c = 151.2 A . A Guinier plot of the X-ray small-angle scattering of the protein solution gave the radius of gyration of the molecule as 27.5 A.

Crystallographic study of endogenous α-amylase inhibitor from wheat

The crystal and molecular structure of a grayanotoxin III derivative, 6-0-acetylgrayanotoxin III is presented. The crystal is orthorhombic, space group P212121, witha=15.582(3),b=21.304(3),c=6.339(3)Å,V=2104(1) Å3Z=4. The structure was solved by direct methods and refined by full matrix least-squares methods to a finalR=0.047 for 1763 independent reflections withF0>3 σ (F0) The molecule is based on a tetracyclic structure consisting of two five-membered, one six-membered, and one seven-membered ring with various conformations.

Ligand-Induced Changes in the Conformation of 3-Isopropylmalate Dehydrogenase from Thermus thermophilus

Abstract Endogenous α-amylase inhibitor from wheat has been crystallized by a microdialysis method. There are two forms of monoclinic crystal in a microdialysis cell with a space group of P 2 1 . The unit cell dimensions are a = 43.5 A , b = 64.8 A , c = 32.2 A , β = 113 ° for the rod-like crystal, and a = 42.5 A , b = 65.2 A , c = 32.2 A , β = 112 ° for the plate-like crystal. The former is suitable for structure analysis because it gives the sharp diffraction beyond 2.0 A resolution, and the latter tends to form a twin crystal. A heavy-atom derivative has been successfully prepared with the heavy-atom reagent K 2 PtCl 4 , and structure analysis is in progress.