The enzymes 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase II (GCHII) catalyze the initial steps of both branches of the bacterial riboflavin-biosynthesis pathway. The structures and molecular mechanisms of DHBPS and GCHII as separate polypeptides are known; however, their organization and molecular mechanism as a bifunctional enzyme are unknown to date. Here, the crystal structure of an essential bifunctional DHBPS/GCHII enzyme from Mycobacterium tuberculosis (Mtb-ribA2) is reported at 3.0 Å resolution. The crystal structure revealed two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. Interestingly, analysis of the crystal packing revealed a long `helical-like oligomer' formed by DHBPS and GCHII functional homodimers, thus generating an `open-ended' unit-cell lattice. However, size-exclusion chromatography studies suggest that Mtb-ribA2 exists as a dimer in solution. To understand the discrepancy between the oligomerization observed in solution and in the crystal structure, the DHBPS (Mtb-DHBPS) and GCHII (Mtb-GCHII) domains of Mtb-ribA2 have been cloned, expressed and purified as His-tagged proteins. Size-exclusion chromatography studies indicated that Mtb-GCHII is a dimer while Mtb-DHBPS exists as a monomer in solution. Moreover, kinetic studies revealed that the GCHII activities of Mtb-ribA2 and Mtb-GCHII are similar, while the DHBPS activity of Mtb-ribA2 is much higher than that of Mtb-DHBPS alone. Taken together, the results strongly suggest that Mtb-ribA2 exists as a dimer formed through its GCHII domains and requires full-length Mtb-ribA2 for optimal DHBPS activity.

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The crystal structure of mesaconic acid, C5H604, has been determined from 491 X-ray intensities, and refined to R = 9.5 %. The crystals are monoclinic with a= 7"225, b = 11.88, c = 7.00 A, ,8= 98°40 ', space group P21/c, Z= 4. The structure consists of infinite chains parallel to a, adjacent molecules being linked by hydrogen bonds of length 2.63 and 2.74 A,. The longer of thcse is a bifurcated hydrogen bond, with its partner of length 2-88 ~ holding the chains together. The C=O bonds in the molecule are in the cis configuration.As part of a programme of studying hydrogen-bonded systems, we have investigated mesaconic acid, also called methylfumaric acid. The present work describes the crystal and molecular structure and adduces evidence to show that the name methylfumaric acid is to some extent misleading, since the C=O bonds are not in the trans configuration. ExperimentalThe crystals were grown from ethanol. They were platy in habit with face (010), and show extinction at an angle of about 16 ° with the long edge [001]. Cell dimensions were obtained from high sin 0 reflexions on Weissenberg films calibrated with silver lincs. The crystal data are a=7"225, b= 11-88, c=7.00 ~, fl=98°40 ', do= 1.44, dc=1.45 g.cm -3, Z=4, space group P21/c, p(Cu K~)= 11"5 cm-491 independent reflexions were collected by Weissenberg photography. The intensities were measured visually and brought to an approximate absolute scale by statistical methods. Determination and refinement of the structureThe structure was solved by interpretation of Patterson projections. The c projection had a marked similarity to one projection of fumaric acid on which one of us (M.P.G.) had worked earlier, and the crystal structure of which is now well established (Brown, 1966; Bednowitz & Post, 1966). Assuming similar linkages of molecules in chains, packing considerations gave reasonable trial coordinates. Full-matrix least-squares refinement (Busing, Martin & Levy, 1962) with unit weights for the observed reflexions and anisotropic thermal factors for carbon and oxygen reduced R to 12.7 %. A three-dimensional difference synthesis served to locate the hydrogen atoms, and a structure factor calculation gave a final R of 9.5 %. The positional and thermal parameters are given in Table l(a) and (b). Observed and calculated structure factors are in Table 2. Bond lengths, bond angles, and intermolecular con-

High-temperature requirement A (HtrA) proteins, which are members of the heat-shock-induced serine protease family, are involved in extracytoplasmic protein quality control and bacterial survival strategies under stress conditions, and are associated with the virulence of several pathogens; they are therefore major drug targets. Mycobacterium tuberculosis possesses three putative HtrAs: HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125). Each has a cytoplasmic region, a transmembrane helix and a periplasmic region. Here, the crystal structure of the periplasmic region consisting of a protease domain (PD) and a PDZ domain from an M. tuberculosis HtrA1 mutant (mHtrA1) is reported at 2.7 Å resolution. Although the mHtrA1 PD shows structural features similar to those of other HtrAs, its loops, particularly L3 and LA, display different conformations. Loop L3 communicates between the PDs of the trimer and the PDZ domains and undergoes a transition from an active to an inactive conformation, as reported for an equivalent HtrA (DegS). Loop LA, which is responsible for higher oligomer formation owing to its length (50 amino acids) in DegP, is very short in mHtrA1 (five amino acids), as in mHtrA2 (also five amino acids), and therefore lacks essential interactions for the formation of higher oligomers. Notably, a well ordered loop known as the insertion clamp in the PDZ domain interacts with the protease domain of the adjacent molecule, which possibly aids in the stabilization of a trimeric functional unit of this enzyme. The three-dimensional structure of mHtrA1 presented here will be useful in the design of enzyme-specific antituberculosis inhibitors.

The cytosolic tryparedoxin peroxidase (cTXNPx) of Leishmania donovani is a defensive enzyme. Apart from the nonsecretory form, the cTXNPx is released in the spent media of Leishmania cultures and also in the host cell cytosol.

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Biological macromolecular assemblies play significant roles in many biological reaction systems, including energy transfer, protein synthesis, protein analysis, DNA replication and signal transduction. More than 10,000 protein structures are now known since the first crystal structure of hemoglobin and myoglobin were determined. On the other hand, only a few macromolecule assembly structures including viruses have been determined by X-ray crystallography. This is because of the difficulties faced in the preparation, crystallization, Xray diffraction measurement, and crystal structure determination of large molecular assemblies. A beamline for biological macromolecular assemblies at SPring-8, which is specially designed to collect high resolution and high quality diffraction data of macromolecule assembly crystals with large unit cell, has been operating since September 1999. We have collected X-ray diffraction intensity data from some crystals of macromolecular assemblies. Diffraction data from a macromolecular assembly crystal with two unique axes of over 600 angstrom has been collected at 3.7 Å resolution. The present status of beamline and some diffraction data will be presented. The Woronin body is a membrane bound organelle, which appears to be restricted to the filamentous Ascomycotina. It is a specialized peroxisome that functions as a plug for the septal pore, in response to cell lysis. Hex1 is the main component protein involved in the in vivo assembly of crystalline Woronin body. The crystal structure of Hex1 at 1.78 Å resolution reveals the intermolecular interactions that promote the Woronin body assembly. The protein contains two mutually perpendicular β -barrels. The N-terminal barrel contains six β -strands and the C-terminal domain contains a five stranded barrel and a flanking α -helix. There are three types of intermolecular interaction. And Groups I and Group II interaction both include the double salt bridges which recruit Hex1 molecular form a coil filament and group III interactions help to crosslink these filaments. Self-assembly is abolished in vitro and in vivo by mutations in intermolecular contact residues, indicating that the crystal structure is a valid representation of the Woronin body-core. In spite of sharing sequence homology, the tertiary structures of Hex1 and the eukaryotic translation initiation factor 5A (eIF-5A) are very similar, suggesting an ancestral link between them. Since eIF-5A does not self-assemble, comparison of these two protein structures suggests how a crystalline protein complex evolved from a soluble precursor. Many Plants produce Ribosome Inactivating Proteins (RIPs)-enzymes that act on ribosomes in a highly specific way, thereby inhibiting protein synthesis. Some RIPs can bind to and enter cells, making them among the most-toxic substances known so far. The lectins from mistletoe belong, like ricin, abrin and modeccin to the group of toxic lectins of A and B chains. The A chain is an enzyme whereas B chain is a lectin. The heterodimeric toxic viscumin was ...

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