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The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 Å. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis is an inflammatory and disabling joint disease affecting 0.5–1.5% of the population. Although various anti-inflammatory (NSAIDs) and disease-modifying (DMARDs) drugs are in clinical use, their precise mechanisms of action are not always defined. In this report, we discuss the effects of widely used DMARDs such as gold derivatives and chloroquine on cathepsins K and S, which have been implicated as critical mediators of inflammation and joint erosion in rheumatoid arthritis. We demonstrate that clinically potent gold derivatives inhibit cathepsins K and S in in vitro and cell-based assays. An X-ray analysis of the gold thiomalate/cathepsin K complex reveals that the inhibitor is bound to the active-site cysteine residue of the protease. Chloroquine, a lysosomotropic agent of lower clinical potency than gold derivatives, inhibits neutral pH-labile cathepsins intracellularly, but does not affect the neutral pH-stable cathepsin S. The potent inhibition of cathepsins implicated in the pathogenesis of rheumatoid arthritis by gold derivatives may explain the therapeutic efficacy of these drugs. [ABSTRACT FROM AUTHOR]

The X-ray crystal structure of the gene product encoded by open reading frame Rv1873 of Mycobacterium tuberculosis has been determined by single isomorphous replacement with anomalous scattering (SIRAS) phasing tech­niques at 1.38 Å resolution from monoclinic crystals with unit-cell parameters a = 33.44, b = 31.63, c = 53.19 Å, β = 90.8°. The 16.2 kDa Rv1873 is a monomer that adopts a primarily α-helical fold with limited structural similarity to previously determined tertiary structures. It has been annotated as a conserved hypothetical protein of unknown function and is classified by the Clusters of Orthologous Groups (COG) database as belonging to COG5579. The three-dimensional structure of the Rv1873 gene product reveals limited similarity to a repeated motif that is found in a variety of other proteins. While not a novel fold, it serves as a model for orthologues predicted to be related by sequence and it is hoped that knowledge of the structure of Rv1873 will aid in determining a possible function for this protein. [ABSTRACT FROM AUTHOR]

The crystal structure of a conserved hypothetical protein corresponding to open reading frame Rv2074 from Mycobacterium tuberculosis ( Mtb) has been solved by the two-wavelength anomalous dispersion method. Refinement of the molecular structure at 1.6 Å resolution resulted in an Rwork of 0.178 and an Rfree of 0.204. The crystal asymmetric unit contains an Rv2074 monomer; however, the crystallographic twofold symmetry operation of space group P43212 generates dimeric Rv2074. Each monomer folds into a six-stranded antiparallel β-barrel flanked by two α-helices. The three-dimensional structure of Rv2074 is very similar to that of Mtb Rv1155, a probable pyridoxine 5′-­phosphate oxidase (PNPOx), which corroborates well with the relatively high sequence similarity (52%) between the two. A structural comparison between Rv2074 and Rv1155 revealed that the core structure (a six-stranded β-barrel) is also well conserved; the major differences between the two lie in the N- and C-­termini and in the small helical domain. Two citric acid molecules were observed in the active site of Rv2074, the crystals of which were grown in 0.2 M sodium citrate buffer pH 5.0. The citric acid molecules are bound to Rv2074 by hydrogen-bonding interactions with Thr55, Gln60 and Lys61. One of the two citric acid molecules occupies the same spatial position that corresponds to the position of the phosphate and ribose sugar moieties of the flavin mononucleotide (FMN) in the Mtb Rv1155–FMN, Escherichia coli PNPOx–FMN and human PNPOx–FMN complex structures. Owing to its extensive structural similarity with Mtb Rv1155 and to the E. coli and human PNPOx enzymes, Rv2074 may be involved in the final step in the biosynthesis of pyridoxal 5′-phosphate (PLP; a vitamin B6). [ABSTRACT FROM AUTHOR]

Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active as an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA. [ABSTRACT FROM AUTHOR]

The X-ray crystal structure of a conserved hypothetical protein of molecular weight 16.3 kDa from Mycobacterium tuberculosis corresponding to open reading frame (ORF) Rv1155 has been solved by the multiwavelength anomalous dispersion method and refined at 1.8 Å resolution. The crystal structure revealed that Rv1155 is a dimer in the crystal and that each monomer folds into a large and a small domain; the large domain is a six-stranded antiparallel β-barrel flanked by two small α-helices and the small domain is a helix–loop–helix motif. The dimer interface is formed by residues protruding primarily from five of the six β-strands in each subunit. Based on structural similarity and on ligand binding, it has been established that Rv1155 is a pyridoxine 5′-phosphate oxidase, the Escherichia coli and human counterparts of which catalyse the terminal step in the biosynthesis of pyridoxal 5′-phosphate (PLP), a cofactor used by many enzymes involved in amino-acid metabolism. The structures of flavin mononucleotide (FMN) and pyridoxal 5′-phosphate (PLP) bound separately to Rv1155 have been determined at 2.2 and 1.7 Å resolution, respectively. Only one monomer binds non-covalently to one FMN molecule or to one PLP molecule. Arg55 and Lys57 are the key residues making hydrogen bonds and ionic inter­actions with the phosphate and ribose groups of the FMN molecule, whereas Arg55 and Arg129 provide hydrogen bonds and ionic interactions with the phosphate group of the PLP. Structural comparisons of Rv1155 from M. tuberculosis with its E. coli and human counterparts demonstrate that the core structure is highly conserved and the FMN-binding site is similarly disposed in each of the structures. [ABSTRACT FROM AUTHOR]

The molecular structure of the pepstatin-insensitive carboxyl peptidase from Scytalidium Iignicolum, formerly known as scytalidopepsin B, was solved by multiple isomorphous replacement phasing methods and refined to an R factor of 0.230 (Rfree = 0.246) at 2.1-Å resolution. In addition to the structure of the unbound peptidase, the structure of a product complex of cleaved angiotensin II bound in the active site of the enzyme was also determined. We propose the name scytalidocarboxyl peptidase B (SCP-B) for this enzyme. On the basis of conserved, catalytic residues identified at the active site, we suggest the name Eqolisin for the enzyme family. The previously uninvestigated SCP-B fold is that of a β-sandwich; each sheet has seven antiparallel strands. A tripeptide product Ala-lle-His, bound in the active site of SCP-B has allowed for identification of the catalytic residues and the residues in subsites S1, S2, and S3, which are important for substrate binding. The most likely hydrolytic mechanism involves nucleophilic attack of a general base (Glu-136)-activated water (OH-) on the si-face of the scissile peptide carbonyl-carbon atom to form a tetrahedral intermediate. Electrophilic assistance and oxyanion stabilization is provided by the side-chain amide of Gin-53. Protonation of the leaving-group nitrogen is accomplished by the general acid function of the protonated carboxyl group of Glu-136. [ABSTRACT FROM AUTHOR]

The crystal structure of the 2A proteinase from human rhinovirus serotype 2 (HRV2-2Apro) has been solved to 1.95 Å resolution. The structure has an unusual, although chymotrypsin-related, fold comprising a unique four-stranded β sheet as the N-terminal domain and a six-stranded β barrel as the C-terminal domain. A tightly bound zinc ion, essential for the stability of HRV2-2Apro, is tetrahedrally coordinated by three cysteine sulfurs and one histidine nitrogen. The active site consists of a catalytic triad formed by His 18, Asp35 and Cys106. Asp35 is additionally involved in an extensive hydrogen-bonding network. Modelling stud- ies reveal a substrate-induced fit that explains the specificity of the subsites S4, S2, S1 and S1′. The structure of HRV2-2Apro suggests the mechanism of the cis cleavage and its release from the polyprotein. [ABSTRACT FROM AUTHOR]

The active site of αlytic protease has been studied with a number of synthetic peptides and compared with similar data published for elastase. The kinetic data indicate that the active site of α-lytic protease extends over at least six subsites (S4-S2). This extended active site has the effect of increasing kcat/Km by more than 106-fold on going from an N-acetylated amino acid amide to a hexapeptide, due mainly to increases in Kcat. There are major differences between α-lytic protease and elastase, both in terms of the kinetic parameters for a number of substrates and in terms of the tertiary structure of the active site. The ability of the S1 subsite to interact with various P1 amino acid side chains differs markedly between the two enzymes, and can be rationalized in terms of the tertiary structural differences. For α-Lytic protease, enzymeesubstratc interactions made in subsite S2 appears to be of primary importance, whereas subsite S4 is most important for elastase. The tertiary structural homology of cz4ytic protease with another bacterial serine protease, Strep tomyces griseus protease A, has allowed detailed model building of a tetrapeptide Ac-Pro-Ala-Pro-Ala-OH at the active site. In this way, the subsites S1 - S4 have been examined for α-lytic protease and compared to other serine proteases. [ABSTRACT FROM AUTHOR]

The gene products of two open reading frames from Mycobacterium tuberculosis ( Mtb) have been crystallized using the sitting-drop vapour-diffusion method. Rv1652 encodes a putative N-acetyl-γ-glutamyl-phosphate reductase ( MtbAGPR), while the Rv1656 gene product is annotated as ornithine carbamoyltransferase ( MtbOTC). Both MtbAGPR and MtbOTC were expressed in Escherichia coli, purified to homogeneity and crystallized. Native data for each crystal were collected to resolutions of 2.15 and 2.80 Å, respectively. Preliminary X-ray data are presented for both enzymes. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis epoxide hydrolases A and B, corresponding to open reading frames Rv3617 and Rv1938, are detoxification enzymes against epoxides. The recombinant forms of these enzymes have been expressed in Escherichia coli and purified to homogeneity. Diffraction-quality crystals of Rv3617 and Rv1938 were obtained by the hanging-drop vapour-diffusion technique. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1 Å resolution, respectively, at the ALS synchrotron at Berkeley, CA, USA. [ABSTRACT FROM AUTHOR]

This analysis of the title compound, C13H13F2IO3, establishes the orientation of ( E)-5-(CH=CH-I) as antiperiplanar ( ap) to the C-C bond (5-6 position) of the 2,4-di­fluoro­phenyl ring system, with the ( E)-5-(CH=CH-I) H atom located in close proximity (2.17 Å) to the F4 atom of the 2,4-di­fluoro­phenyl moiety. [ABSTRACT FROM AUTHOR]

This analysis establishes the rotameric orientation of the pyridyl-ring N atom of the title compound, C17H21N3O4·0.5C6H6, as antiperiplanar ( ap) to the 1,4-dihydropyridine H-4, the absence of an intramolecular hydrogen bond between the 1,4-dihydropyridine NH and the pyridyl-N atom, and the unusual planarity of the 1,4-dihydropyridine ring. [ABSTRACT FROM AUTHOR]