Structure of d(CACGTG), a Z-DNA hexamer containing AT base pairs.

The left-handed Z-DNA conformation has been observed in crystals made from the self-complementary DNA hexamer d(CACGTG). This is the first time that a non disordered Z form is found in the crystal structure of an alternating sequence containing AT base pairs without methylated or brominated cytosines. The structure has been determined and refined to an agreement factor R = 22.9% using 746 reflections in the resolution in the resolution shell 7 to 2.5 A. The overall shape of the molecule is very similar to the Z-structure of the related hexamer d(CG)3 confirming the rigidity of the Z form. No solvent molecules were detected in the minor groove of the helix near the A bases. The disruption of the spine of hydration in the AT step appears to be a general fact in the Z form in contrast with the B form. The biological relevance of the structure in relation to the CA genome repeats is discussed.     

Crystallization,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

The current situation of health research and ethics in Sudan

At the beginning of the twentieth century, health research in the Sudan developed primarily as a function of the colonial British administration. Ethical issues in the medical profession in the Sudan are governed by the Sudan Medical Council. To address these issues, the Sudan Medical Council issued Medico-legal and Ethical Guidelines in 1967. This important document has focused principally on ethical issues arising in clinical medicine. Throughout the history of health research in Sudan it is very difficult to find any reference to research ethics. Nevertheless, there have been a few attempts to articulate ethics in health research. In 1979, Sudan witnessed the establishment of the first ethical review committee, which was established by the initiative of a group of doctors and scientists from the national health research laboratory. This committee got neither political nor institutional recognition. Therefore, it was not developed and came to an end shortly after its inception. Yet, in 2000 the FMOH established an ethical review committee (ERC). The functions of this committee were to review proposals of health research to be carried out in the country for ethical issues irrespective to the funding agents. Unfortunately, the committee devoted all the jobs in reviewing only externally-sponsored health research and research carried out by expatriate researchers or international organisations.     

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7A resolution

The catalase-peroxidase encoded by katG of Burkholderia pseudomallei (BpKatG) is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid as an antibiotic. The structure of a complex of BpKatG with an unidentified ligand, has been solved and refined at 1.7A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are 15.3% and 18.6%, respectively. The crystallized enzyme is a dimer with one modified heme group and one metal ion, likely sodium, per subunit. The modification on the heme group involves the covalent addition of two or three atoms, likely a perhydroxy group, to the secondary carbon atom of the vinyl group on ring I. The added group can form hydrogen bonds with two water molecules that are also in contact with the active-site residues Trp111 and His112, suggesting that the modification may have a catalytic role. The heme modification is in close proximity to an unusual covalent adduct among the side-chains of Trp111, Tyr238 and Met264. In addition, Trp111 appears to be oxidized on C(delta1) of the indole ring. The main channel, providing access of substrate hydrogen peroxide to the heme, contains a region of unassigned electron density consistent with the binding of a pyridine nucleotide-like molecule. An interior cavity, containing the sodium ion and an additional region of unassigned density, is evident adjacent to the adduct and is accessible to the outside through a second funnel-shaped channel. A large cleft in the side of the subunit is evident and may be a potential substrate-binding site with a clear pathway for electron transfer to the active-site heme group through the adduct.     

Retinoic acid binds to the C2-domain of protein kinase C(alpha)

Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.     

A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase

Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 A and 2.5 A resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha(3)beta(8)alpha(4) sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the beta sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta(5)beta(4) sandwich fold and three alpha helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate.     

Assessment of the ethical review process in Sudan

The ethical review process is an important component of contemporary health research worldwide. Sudan started an ethical review process rather late in comparison with other countries. In this study, we evaluate the structure and functions of existing ethics review committees. We also explore the knowledge and attitudes of Sudanese researchers toward the ethical review process and their experience with existing ethics review committees. There are four ethics review committees in the country; these committees have no institutional regulations to govern their functions. Furthermore, Sudan also lacks national guidelines. Ethical reviews are carried out primarily for studies seeking international funding and are almost always governed by the funding agencies' requirements. Nearly half of respondents (46.3%) knew about the existence of research ethics committees in Sudan. Researchers reported a variety of experiences with the ethical review process; most of them were unable to define 'ethics committee'.     

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.     

Structural characterization of the Ser324Thr variant of the catalase-peroxidase (KatG) from Burkholderia pseudomallei

The Ser315Thr variant of the catalase-peroxidase KatG from Mycobacterium tuberculosis imparts resistance to the pro-drug isonicotinic acid hydrazide (isoniazid) through a failure to convert it to the active drug, isonicotinoyl-NAD. The equivalent variant in KatG from Burkholderia pseudomallei, Ser324Thr, has been constructed, revealing catalase and peroxidase activities that are similar to those of the native enzyme. The other activities of the variant protein, including the NADH oxidase, the isoniazid hydrazinolysis and isonicotinoyl-NAD synthase activities are reduced by 60-70%. The crystal structure of the variant differs from that of the native enzyme in having the methyl group of Thr324 situated in the entrance channel to the heme cavity, in a modified water matrix in the entrance channel and heme cavity, in lacking the putative perhydroxy modification on the heme, in the multiple locations of a few side-chains, and in the presence of an apparent perhydroxy modification on the indole nitrogen atom of the active-site Trp111. The position of the methyl group of Thr324 creates a constriction or narrowing of the channel leading to the heme cavity, providing an explanation for the lower reactivity towards isoniazid and the slower rate of isonicotinoyl-NAD synthesis.