Crystallization and preliminary x-ray diffraction study of the flavoprotein NADH peroxidase from Streptococcus faecalis 10C1

NADH peroxidase from Streptococcus faecalis 10C1 has been crystallized from ammonium sulfate solutions using the hanging drop vapor diffusion method. Depending on pH, the crystals grew in the orthorhombic space group I222 or one of its subgroups P222 or P2(1)2(1)2 (or one of its two permutations). In both cases the unit cell axes are a = 76.6 A, b = 132.9 A, and c = 145.7 A. There are two monomers/asymmetric unit in the body-centered crystal form and four in the primitive one. The enzyme is catalytically active in the crystalline state. The crystals diffract to at least 2.5 A resolution; they are stable in the x-ray beam and hence suitable for detailed three-dimensional structure determination.     

Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes

We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NOx) analyzer for the quantitation of any nitric oxide present in these gases. The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013. In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations. The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques.     

One thousand heart attacks in Grampian: the place of cardiopulmonary resuscitation in general practice.

The outcome of 1011 heart attacks in patients under the care of general practitioners who practised cardiopulmonary resuscitation and were equipped with defibrillators is reported. The 28 day mortality was 36% (367 patients), and 59% of deaths occurred outside hospital. The general practitioner was the first medical contact in 92% of heart attacks and was equipped with a defibrillator in 80% of such calls. Fifty six patients had a cardiac arrest in the presence of a general practitioner, and resuscitation was attempted in 47 cases, representing 5% of all calls for heart attacks. Twenty one (45%) resuscitated patients reached hospital alive, and 13 (28%) survived to leave hospital. The opportunities for cardiopulmonary resuscitation in general practice occur sufficiently often to warrant training and equipping general practitioners for advanced life support. The results of resuscitation by general practitioners working alone compare favourably with those of mobile coronary care units based in hospitals.     

Periplasmic secretion of human growth hormone by Escherichia coli

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.     

A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X-ray crystallography at 0.3-nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione-I is bound in a V-shaped conformation, while glutathione-II is extended. The zwitterionic glutamyl end of glutathione-II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione-I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58-S gamma. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.     

Microbial transformation of a fungicide-derived amine, dimethyl-p-phenylene diamine by Alcaligenes DM4

Abstract Microbial transformation of N , N -dimethyl- p -phenylene diamine (DMPDA), a microbial product formed from the fungicide fenaminosulf ( p -dimethylaminobenzenediazo sodium sulfonate) was studied by enriching microbes in soils treated with the amine. Microorganisms isolated from DMPDA-treated soil belonged to the genera of Micrococcus, Alcaligenes , and Corynebacterium . Of the various isolates, Alcaligenes DM₄ showed maximal growth on DMPDA utilizing it as sources of carbon and nitrogen. When grown in mineral salts basal medium containing 0.05% DMPDA to serve as carbon and nitrogen sources, Alcaligenes DM₄ grew exponentially up to 18 h. Even though the characterization of the complete pathway of microbial degradation of DMPDA could not be carried out due to the auto-oxidation of the compound, the initial transformation product of DMPDA by Alcaligenes DM₄ has been identified as a dimer. The dimer is generated into the culture medium presumably by the extra-cellular oxidase of Alcaligenes DM₄. It is suggested that the risk-benefit evaluation on the use of fenaminosulf is to be made taking into consideration the microbial transformations of the fungicide.     

Purification, crystallization, and preliminary x-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.     

Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.

The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.