Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes.

Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (ßF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: ßF41Y; ßK66Y; ßF71Y; ßT84Y; ßF85Y; and ßL96Y. As with ßF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (ßT84Y, and ßF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in ßT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 µM ascorbate over a time period of 60 min increasing from 10% in ßF41Y to over 50% in ßT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

The biochemical and molecular spectrum of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTCase) deficiency, the most common inherited urea cycle disorder, is transmitted as an X-linked trait. The clinical phenotype in affected males as well as heterozygous females shows a spectrum of severity ranging from neonatal hyperammonaemic coma to asymptomatic adults. The ornithine transcarbamylase enzyme is a trimer with three active sites per holoenzyme molecule, each of which is composed of an interdomain region of one polypeptide and a polar domain of the adjacent polypeptide. The OTC gene is located on the short arm of the X-chromosome and one of the two alleles undergoes inactivation in female cells. Approximately 140 mutations have been found in families affected with OTCase deficiency, most having their own 'private' mutation. Large deletions of one exon or more are seen in approximately 7% of patients, small deletions or insertions are seen in about 9%, and the remaining mutations are single base substitutions. Approximately 15% of mutations affect RNA splicing sites. The recurrent mutations are distributed equally among CpG dinucleotide hot spots. Generally, mutations causing neonatal disease affect amino acid residues that are 'buried' in the interior of the enzyme, especially around the active site, while those associated with late onset and milder phenotypes tend to be located on the surface of the protein. Very few mutations have been found in the sequence of the leader peptide, proportionally much fewer than in the sequence of the mature enzyme. Only few of the mutations have been expressed in bacteria or mammalian cells for the study of their deleterious mechanisms. Examples of expressed mutations include R277W and R277Q associated with late-onset disease, which markedly increase the Km for ornithine, shift the pH optimum to more alkaline and decrease the thermal stability of the purified mutant enzyme. R141Q (neonatal disease) disrupts the active site, whereas the purified R40H mutant has normal catalytic function and this mutation is likely to affect posttranslational processing such as mitochondrial targeting. It appears that most new mutations occur in male sperm and are then passed on to a transmitting heterozygous female. Uncommonly, mild mutations are transmitted by asymptomatic males to their daughters, subsequently resulting in clinical disease of males in future generations. The causes for variable expressivity of these mutations are currently unknown but are likely to involve a combination of environmental and genetic modifiers.

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains.

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

Hepatic glutamine synthetase deficiency in fatal hyperammonemia after lung transplantation.

BACKGROUND: The cause of severe acquired hyperammonemia, an uncommon but often fatal complication of organ transplantation and chemotherapy for cancer, is obscure. OBJECTIVE: To test the hypothesis that liver glutamine synthetase deficiency may explain hyperammonemia in patients who have had organ transplantation or are receiving chemotherapy. DESIGN: Case report. PATIENTS: Two patients who had fatal hyperammonemia after orthotopic lung transplantation. MEASUREMENTS: Liver tissue was analyzed to determine the activities of two urea cycle enzymes and glutamine synthetase. Western blot assays for hepatic glutamine synthetase were performed to determine whether glutamine synthetase deficiency resulted from reduced enzyme levels. RESULTS: Activities of carbamoyl phosphate synthetase I and ornithine carbamoyltransferase in the liver were normal. The activity of hepatic glutamine synthetase was markedly reduced (in patient 1, 12% of the mean value in controls; in patient 2, 28% of the mean value in controls), and a concomitant reduction in the amount of glutamine synthetase protein was observed. CONCLUSION: Hyperammonemia after transplantation was associated with hepatic glutamine synthetase deficiency in two patients, but the causal relation between these two conditions must be further studied.

Identification of 'private' mutations in patients with ornithine transcarbamylase deficiency.

The majority of cases of ornithine transcarbamylase deficiency are due to novel mutations making it impossible to develop common methods for genetic analysis. However, identification of causative mutations has important implications for diagnosis (particularly prenatal diagnosis), prediction of likely course and outcome and the eventual possibility of gene therapy. As part of a continuing study of ornithine transcarbamylase deficiency, we now report an additional thirty novel mutations in the ornithine transcarbamylase gene, together with a brief summary of their clinical presentations.

'Late onset' ornithine transcarbamylase deficiency: function of three purified recombinant mutant enzymes.

Although many mutations in the ornithine transcarbamylase gene have been correlated with 'late onset' of hyperammonemia in patients, the effects of these mutations on enzyme function are largely unknown. Three recurrent mutations (R40H, R277W and R277Q) found in patients with 'late onset' disease were incorporated into 'mature' human ornithine transcarbamylase cDNA and overexpressed in Escherichia coli. The three recombinant mutant enzymes were purified to homogeneity on an affinity column and their biochemical characteristics were compared to the wild type enzyme. The R277W and R277Q mutants display markedly reduced affinity for L-ornithine, loss of substrate inhibition, alkaline shift of pH optimum, and reduced thermal stability compared to the wild type enzyme. These differences, particularly the reduced affinity for L-ornithine, are sufficient to account for their biochemical effects. In contrast, the 'mature' R40H mutant was biochemically indistinguishable from the wild type enzyme in vitro.

Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces 'late onset' hyperammonaemia.

Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders. Approx. 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients [Tuchman (1993) Hum. Mutat. 2, 174-178; Tuchman and Plante (1995) Hum. Mutat. 5, 293-295]. A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase) [Tuchman, Morizono, Reish, Yuan and Allewell (1995) J. Med. Genet. 32, 680-688], and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase [Villeret, Tricot, Stalon and Dideberg (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10762-10766], indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein. However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coli aspartate transcarbamylase, comprised of residues 230-250) of ATCase. Human wild-type OTCase and the R277W mutant have been cloned and overexpressed in E. coli and a rapid and efficient purification method utilizing the bisubstrate analogue, Ndelta-(phosphonacetyl)-L-ornithine, has been developed and used to purify both proteins. Gel chromatography indicates both are trimeric. The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E. coli OTCase [Kuo, Herzberg and Lipscomb (1985) Biochemistry 24, 4754-4761], suggesting that its catalytic mechanism is similar, although its maximal activity is approx. 10-fold less. Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for L-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5 degrees C. Its reduced affinity for L-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces.

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.

Expression, purification, and characterization of recombinant human glutamine synthetase.

A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx. 8 mg of purified enzyme per litre of culture. The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized. It has a subunit molecular mass of approx. 45000 Da. The Vmax value obtained using a radioactive assay with ammonia and l-[G-3H]glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate. Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2. 9 mM for the radioactive and spectrophotometric assays respectively. The Km for ammonia in the radioactive assay was 0.15 mM. The midpoint of thermal inactivation was 49.7 degrees C. Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation. The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography.

Electron microscopy of Bacillus subtilis GroESL chaperonin and interaction with the bacteriophage phi 29 head-tail connector.

The Bacillus subtilis GroESL chaperonin was isolated by sucrose density gradient centrifugation and the constituent GroES and GroEL moieties were purified by electrophoresis in agarose. Electron microscopic images of negatively stained GroEL and GroES oligomers and GroESL complexes were averaged using a reference-free alignment method. The GroEL and GroES particles had the sevenfold symmetry characteristic of their Escherichia coli counterparts. GroESL complexes, reconstituted efficiently in vitro from GroEL and GroES in the absence of added ADP or ATP, had the characteristic bullet- and football-like shapes in side view. Purified bacteriophage phi 29 head-tail connectors having a mass in excess of 0.4 MDa were shown to bind to GroESL at the end opposite to the GroES. The same GroESL-connector complexes were isolated from phage-infected cells in which capsid assembly was blocked, and thus the complex may have functional significance in phi 29 morphogenesis.

Purified Pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages.

We obtained biologically active purified leukotoxin (Lkt) from Pasteurella haemolytica serotypel, strain 12296 using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three species of Lkt of molecular masses 95, 100, and 104 kDa were obtained. Purity of all three species of Lkt was confirmed by analytical SDS-PAGE and Western blot (immunoblot) analysis. Results from the chromogenic Limulus amebocyte lysate assay and silver staining of SDS-PAGE patterns indicated that the preparations were free of contaminating lipopolysaccharide. We then studied the kinetics of TNF alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNF alpha and IL-1 beta gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNF alpha and IL-1 beta mRNA expression were detectable at 1 h after stimulation with 1 leukotoxin unit/ml. The expression peaked at 2 h, steadily declining up to 6 h, and was undetectable by 10 h. Secreted TNF alpha measured by bioassay peaked at 4-6 h and accumulated at a lesser concentration after 6 h. By contrast, secreted IL-1 peaked at 6 h and decreased significantly by 10 h. The ability of purified Lkt to induce TNF alpha and IL-1 beta gene expression and secretion of bioactive proteins was suppressed by Ca2+ chelating agents, 5 mM EDTA and 5 mM EGTA, but not polymyxin B. Heat-inactivation of the purified Lkt that had lost its cytocidal property completely abrogated induction of TNF alpha and IL-1 beta gene expression and secretion in bovine AMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.

sRNA of phage phi 29 of Bacillus subtilis mediates DNA packaging of phi 29 proheads assembled in Escherichia coli.

The structural genes of the prohead of phage phi 29 of Bacillus subtilis and a small phi 29 RNA (sRNA) were cloned and expressed in Escherichia coli individually or in combination to study the role of the sRNA in prohead assembly and the mechanism of prohead morphogenesis. The genes coding for the proteins of the scaffold (gp7), the capsid (gp8), the portal vertex (gp10), and the dispensable head fiber (gp8.5) were expressed in E. coli and the gene products were assembled, with and without the presence of the sRNA, into uniform and prolate particles that resembled the typical native phi 29 prohead. No differences in particle size and shape were found between the particles of 7-8-8.5-10 (scaffold-capsid-fiber-portal vertex) and 7-8-8.5-10-RNA (scaffold-capsid-fiber-portal vertex-RNA), suggesting that the phi 29 sRNA was not required for phi 29 prohead assembly. The 7-8-8.5-10 particles produced in E. coli in the absence of phi 29 sRNA were fully competent to package phi 29 DNA in the defined in vitro DNA packaging system by the addition of purified sRNA. Moreover, these DNA-filled heads were assembled into infectious virions in extracts. Without the addition of the sRNA, the 7-8-8.5-10 particles were incompetent while the 7-8-8.5-10-RNA particles were competent in DNA packaging. Bacterial sRNA present in E. coli cannot substitute for the phi 29 sRNA. The assembly of prohead particles in E. coli indicated that host factors unique to B. subtilis were not required. The evidence that the phi 29 sRNA was not required for phi 29 prohead assembly and was not a fixed structural component of the phi 29 prohead favors the conclusion that the phi 29 sRNA is a specific enzyme or morphogenetic factor in DNA packaging.

Thiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world.

Among sulfur compounds, thiosulfate and polythionates are present at least transiently in many environments. These compounds have a similar chemical structure and their metabolism appears closely related. They are commonly used as energy sources for photoautotrophic or chemolithotrophic microorganisms, but their assimilation has been seldom studied and their importance in bacterial physiology is not well understood. Almost all bacterial strains are able to cleave these compounds since they possess thiosulfate sulfur transferase, thiosulfate reductase or S-sulfocysteine synthase activities. However, the role of these enzymes in the assimilation of thiosulfate or polythionates has not always been clearly established. Elemental sulfur is, on the contrary, very common in the environment. It is an energy source for sulfur-reducing eubacteria and archaebacteria and many sulfur-oxidizing archaebacteria. A phenomenon still not well understood is the 'excessive assimilatory sulfur metabolism' as observed in methanogens which perform a sulfur reduction which exceeds their anabolic needs without any apparent benefit. In heterotrophs, assimilation of elemental sulfur is seldom described and it is uncertain whether this process actually has a physiological significance. Thus, reduction of thiosulfate and elemental sulfur is a common but incompletely understood feature among bacteria. These activities could give bacteria a selective advantage, but further investigations are needed to clarify this possibility. Presence of thiosulfate, polythionates and sulfur reductase activities does not imply obligatorily that these activities play a role in thiosulfate, polythionates or sulfur assimilation as these compounds could be merely intermediates in bacterial metabolism. The possibility also exists that the assimilation of these sulfur compounds is just a side effect of an enzymatic activity with a completely different function. As long as these questions remain unanswered, our understanding of sulfur and thiosulfate metabolism will remain incomplete.

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.

Methanogenic bacteria from human dental plaque.

Samples of human dental plaque were examined for the presence of methanogenic bacteria. Of 54 samples from 36 patients, 20 yielded H2/CO2-using methanogenic enrichment cultures. All methanogen-positive samples were from patients with some degree of periodontal disease. The predominant populations in the enrichments had morphologies characteristic of Methanobrevibacter spp. In six enrichments derived from three patients, the common methanogen was antigenically similar to Methanobrevibacter smithii. The same was true for the three methanogenic isolates obtained in axenic culture from a fourth patient. The six enrichments and two of the three isolates were antigenically closer to strain ALI than to PS. Two of the enrichments also had subpopulations with weak antigenic similarity to Methanosphaera stadtmanae. The data indicate that methanogens in the oral cavity of humans are antigenically close to those found in the intestinal tract.

Bacterial Methanogenesis and Growth from CO2 with Elemental Iron as the Sole Source of Electrons.

Previous studies of anaerobic biocorrosion have suggested that microbial sulfur and phosphorus products as well as cathodic hydrogen consumption may accelerate anaerobic metal oxidation. Methanogenic bacteria, which normally use molecular hydrogen (H(2)) and carbon dioxide (CO(2)) to produce methane (CH(4)) and which are major inhabitants of most anaerobic ecosystems, use either pure elemental iron (Fe(0)) or iron in mild steel as a source of electrons in the reduction of CO(2) to CH(4). These bacteria use Fe(0) oxidation for energy generation and growth. The mechanism of Fe(0) oxidation is cathodic depolarization, in which electrons from Fe(0) and H(+) from water produce H(2), which is then released for use by the methanogens; thermodynamic calculations show that significant Fe(0) oxidation will not occur in the absence of H(2) consumption by the methanogens. The data suggest that methanogens can be significant contributors to the corrosion of iron-containing materials in anaerobic environments.

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria.

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.