Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

Some metabolic relationships between biopterin and folate: Implications for the “methyl trap hypothesis”

Tetrahydrobiopterin and the folate coenzymes can reciprocally interact in ways that would be useful to the metabolic pathways subserved by both of these coenzymes. Thus, through one of the reactions catalyzed by methylene tetrahydrofolate reductase, 5-CH₃-H₄-folate can regenerate BH₄ from q-BH₂ and q-BH₂ can provide an escape from the methyl trap.Special issue dedicated to Dr. Louis Sokoloff     

Distribution of nitrate assimilation between the root and shoot of legumes and a comparison with wheat

Legumes of the Phaseoleae ( Glycine max L. Merr., Phaseolus coccineus L., P. vulgaris L., Vigna radiata L. Wilczek and V. unguiculata L. Walp.), when grown on 10 m M nitrate, had a low in vitro nitrate reductase (NR) activity in the root compared to the shoot (<15%). In legumes of the Vicieae ( Cicer aerietinum L., Pisum sativum L. and Vicia faba L.), Genisteae ( Lupinus albus L.) and Trifolieae ( Medicago sativa L. and M. truncatula Gaertn.), 30–60% of their total NR activity was in the root. The Phaseoleae had a higher nitrate content in the shoot. Decreasing the nitrate supply increased the relative proportion of NR activity in the root of garden pea ( Pisum sativum ) and wheat but did not alter the predominantly leaf-based assimilation of nitrate in Phaseolus vulgaris. When in vitro NR activity of the pea shoot was compared with the in vivo NR activity and the rate of accumulation of reduced N by this tissue, similar values were obtained. In vitro NR activity of the wheat shoot was 5 times its in vivo NR activity and 12 times its rate of accumulation of reduced N.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Influence of partial defoliation of green pepper on the senescence,growth, and nitrate reductase of the remaining leaf

Summary Removal of all but one leaf from pepper plants prevented the senescence of the remaining leaf and caused increases of approximately 140, 200, and 200%, respectively in leaf area, weight, and nitrate reductase activity. Development of the fruit (fresh and dry weight increases) was only approximately 65% of that of fruit on control plants.     

Effects of seasonal changes of soil salinity and soil nitrogen on the N-metabolism of the halophyteArthrocnemum fruticosum (L.) Moq.

Summary The N-metabolism ofArthrocnemum fruticosum (L.) Moq., growing in a saline area north-east of the Dead Sea in Jordan, was studied over its vegetative growth period from March to September 1981. Plant and soil samples were taken at monthly intervals. Water content, Na⁺, K⁺, Cl⁻, NH ₄ ⁺ , NO ₂ ⁻ and NO ₃ ⁻ concentrations were determined in the soil extracts, and the same determinations plus ash weight, soluble carbohydrates, proline, proteins andin vivo nitrate reductase in the plant roots and shoots. Soil humidity decreased and salinity increased from March to August, with re-wetting occurring in late July. K⁺ and Cl⁻ were much lower in the soils than Na⁺. Plant relative dry weight increased during summer due to the absorption of Na⁺ in addition to increased organic dry weight. The uptake of Na⁺ was not balanced by a similar uptake of Cl⁻. Ammonium and nitrate decreased in soil and plants in parallel with increasing salinity. Nitrite was only found in the roots and always in very low quantities. Proline was found only in March. The total soluble carbohydrates in the roots showed a short increase in June when the sodium in the plants also increased. It was concluded that carbohydrates may be used to balance osmotic shocks, but that another compatible compounds is necessary to maintatin long-term osmotic equilibrium. The nitrate reductase activity, measuredin vivo, and the soluble protein changed roughly in parallel with the internal nitrate from May to August, suggesting that nitrogen uptake and reduction in the plant is inhibited during summer when the soil is dry and very saline. This could be a direct effect of drought and/or salinity on the plants, or an indirect onevia an inhibition of nitrifying bacteria.     

Amino acid sequence of COOH-terminal 20K Da fragment from pig liver microsomal NADPH-cytochrome P-450 reductase

We have determined the complete amino acid sequence of a 20K Da COOH-terminal fragment of porcine NADPH-cytochrome P-450 reductase. The 20K Da fragment is probably produced by a proteolytic cleavage of the intact protein in porcine liver microsomes, and since the cleavage does not affect enzymatic activity, the fragment has been studied as a distinct domain. The sequence comprises 175 amino acids including three cysteine residues, one of which has been previously identified as protected by NADPH from S-carboxymethylation. The NADPH-protected cysteine lies in a stretch of 12 residues with partial homology to glutathione reductase, and is adjacent to a hydrophobic region containing a glycine-rich stretch homologous to other FAD-containing proteins. The predicted secondary structure over this entire region is beta-sheet/beta-turn/beta-sheet/alpha-helix/beta-sheet/beta-turn/alpha-h elix corresponding to hydrophobic residues 21-28/glycine-rich residues 29-33/residues 34-38/residues 39-54/residues 56-61/NADPH-protected cysteine residues 62-78/residues 71-82. It is possible that the 20K Da domain provided a significant portion of the sequence responsible for binding FAD and NADPH in the intact enzyme. This data provides a basis for further active site studies.     

The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley

The effect of cadmium on the biosynthesis of chlorophyll has been investigated in the leaves of dark-grown seedlings of barley ( Hordeum vulture L. cv. Proctor). Cd2+ inhibited the production of chlorophyll by affecting 1) the synthesis of 5-aminolacvulinic acid and 2) the protoehlorophyllide reductase ternary complex with its substrates. Cd2+ had no effect on the constituent enzymes that catalyse the synthesis of free protoehlorophyllide from 5-aminolaevulinic acid. The results obtained are consistent with Cd2+ inhibiting the formation of chlorophyll by reacting with essential thiol groups in both the protochlorophyllide reductase protein and the enzyme(s) involved in the light dependent synthesis of 5-aminolaevulinic acid.