Biochemical characterization of the molybdenum cofactor mutants of Neurospora crassa: in vivo and in vitro reconstitution of NADPH-nitrate reductase activity

Summary Molybdenum cofactor (MoCo) mutants of Neurospora crassa lack both NADPH-nitrate reductase and xanthine dehydrogenase activity. In vivo and in vitro studies to further characterize these mutants are now reported. The MoCo mutants nit-9A and nit-9B are capable of growing, albeit poorly, with nitrate as the sole nitrogen source, provided high levels of molybdate are present. The MoCo mutants nit-9A, nit-9B and nit-9C, but not nit-1, nit-7 or nit-8, have significant levels of NADPH-nitrate reductase when grown in nitrate medium containing 30 mM molybdate. In vitro reconstitution experiments using cell free extracts of the N. crassa MoCo mutants and E. coli HB101 as a source of wild-type MoCo were performed. MoCo from E. coli was capable of reconstituting NADPH-nitrate reductase activity to nit-1, nit-7 and nit-8. Molybdate is required for the in vitro reconstitution of NADPH-nitrate reductase activity. It was not possible to in vitro reconstitute NADPH-nitrate reductase activity in the MoCo mutants nit-9A, nit-9B or nit-9C.     

Genetic analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardii

Summary Six mutants (305, 301, 203, 307, 104 and 102) of Chlamydomonas reinhardii, all defective in nitrate reductase (NR) activity, have been genetically analyzed. All except 102 carry single Mendelian mutations.Mutant 305, defective in diaphorase activity and mutant 301, defective in terminal enzyme activity, did not give rise to wild-type recombinants when crossed to each other or with the nit-1 mutant isolated from strain 137c (which is actually a double mutant nit-1 nit-2). Nit-1 was shown to lack both diaphorase and terminal activities. Whether the mutated sites in 305 and 301 are located in a unique cistron (nit-1) or in two adjacent cistrons (nit-1a and nit-1b) coding for a diaphorase subunit and a terminal subunit of NR is discussed in the light of previous biochemical findings.The 203 mutation affecting a regulatory gene did not recombine with nit-2, the other mutated locus present in strain 137c.Mutants 307, 104 and 102, all lacking molybdenum cofactor for both NR and xanthine dehydrogenase, where shown to be affected in different loci. The genes mutated in 307 and 104 have been designated nit-3 and nit-4, respectively. The 102 strain is mutated in two non-linked loci, nit-5 and nit-6, with both mutations required to confer the mutant phenotype. One of these cryptic mutations is present in the wild strain 21gr.The results indicate that at least six or seven loci are involved in the production of an active NR enzyme: one (nit-1) or two (nit-1a and nit-1b) cistrons to produce the NR apoproteins responsible for the partial activities diaphorase and terminal, one locus (nit-2) for the regulation of NR synthesis, and four loci (nit-3, nit-4, nit-5 and nit-6) to produce the molybdenum cofactor. The loci nit-1a and nit-2 seem to correspond to the nit-A and nit-B loci described by Nichols and Syrett (J Gen Microbiol 108:71–77, 1978).Abbreviations NR nitrate reductase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - MoCo molybdenum-containing cofactor - PD parental ditype - NPD non-parental ditype - TT tetratype - WT wild type - BVH reduced benzyl viologen     

Cloning and preliminary characterization of a molybdenum cofactor gene of Neurospora crassa

Summary A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli ch1D molybdenum cofactor mutants (ch1D, ch1D::Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band of N. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants (ch1A, ch1B, ch1C, ch1D, ch1E, ch1G and ch1M), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the ch1D mutant. In vitro reconstitution experiments using wild-type, ch1D and ch1D; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. crassa MoCo mutants nit-1, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from ch1D; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original ch1D mutant.     

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa

Summary A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.     

Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa

Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.     

The ftr cistron of Coprinus cinereus is the structural gene for a multifunctional transport carrier molecule

Summary Mutants of the basidiomycete Coprinus cinereus which were selected for their resistance to growth inhibitions caused by hexose analogues are all alleles of the ftr cistron. They are shown to have approximately normal levels of activity of enzymes involved in intracellular sugar metabolism and to accumulate normal levels of sugar phosphates. However, the mutants show greatly depressed rates of sugar uptake. Uptake rates from 0.01 mM solutions of 2-deoxy-D-glucose were only 1 to 4% of the wild type rate, and from 15 mM solutions the mutant rates were between 16 and 40% of normal. Kinetic analysis showed that the mutant V_max values were reduced to a few per-cent of normal while K_m values were relatively little changed and in some cases the mutants had an increased affinity for the substrate. Reverse mutations restored the V_max value and the K_m to about the wild type level. Previous data had shown that position of mutants within the allele map depended on selection conditions in a way that implied some interaction between the ftr gene product and the substrate. Since the mutants are defective in transport from both high and low sugar concentrations, and since they exhibit coordinated alterations in K_m and V_max, it is concluded that the ftr cistron is the structural gene for a product involved in sugar translocation (both as carrier and energisation link) in both high and low affinity glucose transport systems.     

A Missense Mutation in the Sodium Channel β2 Subunit Reveals SCN2B as a New Candidate Gene for Brugada Syndrome

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%–25% of BrS patients carry genetic defects that cause loss‐of‐function of the voltage‐gated cardiac sodium channel. Thus, 70%–75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (I_Na) from cells coexpressing Na_v1.5 and wild‐type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in I_Na density when Na_v1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Na_v1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Na_v1.5 cell surface expression. The effect of the mutant β2 subunit on the I_Na strongly suggests that SCN2B is a new candidate gene associated with BrS.     

Mutational and Pharmacological Alterations of Neuronal Membrane Function Disrupt Conditioning in Drosophila

Neuronal membrane channels of Drosophila melanogaster were altered either genetically or pharmacologically in order to investigate the role of specific ionic currents in the acquisition and retention of a conditioned behavior. Conditioning could not be detected for Shaker mutants, in which the fast transient potassium current (I_A) is altered; a second potassium channel mutant, eag (ether a go-go) is conditioned like wild type, but the retention period is abnormally short. The nap^ts mutant (no action potential, temperature sensitive), in which nerve excitability is reduced, also expresses normal acquisition and a shortened period of retention. Double mutants of Sh⁵ and nap^ts as well as Sh⁵ treated with tetrodotoxin, are essentially normal with respect to acquisition; in both cases these flies remain retention-defective. These experiments therefore reveal a behavioral phenotype of Drosophila mutants in which the primary physiological defect seems to be in the functioning of specific neuronal ionic channels.     

Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number,growth rate and enzyme activity

Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.     

Two distinct calcium-activated potassium currents in larval muscle fibres ofDrosophila melanogaster

The non-synaptic membrane currents of muscle fibres have been studied in late embryogenesis ofDrosophila melanogaster using the voltage-clamp technique in wild-type andShaker mutant third instar larvae. Five currents were found in the wild type muscle membrane at this embryonic stage: one fast inward Ca current (I_Ca), two fast outward K currents (I_A and I_Acd) and two slow outward K currents (I_K and I_C). I_Acd and I_C are Ca-dependent.Several procedures were used to separate I_Acd from I_A: I_Acd is present inShaker mutants which are characterized by the absence of I_A (Salkoff and Wyman 1981); I_Acd, but not I_A, is suppressed by Co²⁺ (10 mM) or La³⁺ (1 mM); I_Acd shows steady-state inactivation at more positive potentials than I_A; I_Acd, unlike I_A, is 3,4-diaminopyridine (3,4-DAP) resistant. Furthermore, tetraethylammonium (TEA, 20 mM) which is known to be uneffective on I_A, blocks I_Acd. I_Acd could not be triggered by using strontium or barium as calcium substitutes. By partial substitution of Ca by Ba or Sr ions, it was found that Ba, but not Sr, blocks the I_Acd channel.A non-inactivating, TEA sensitive, Ca-dependent K current (I_C), which gave N-shaped I-V plots, could be separated from I_K by using Ca-channel blockers. I_C and I_K activate at membrane potentials of about –25 mV and –10 mV, respectively.The participation of I_Acd and I_C to membrane electrophysiology is discussed.     

Identical NR5A1 Missense Mutations in Two Unrelated 46,XX Individuals with Testicular Tissues

The role of monogenic mutations in the development of 46,XX testicular/ovotesticular disorders of sex development (DSD) remains speculative. Although mutations in NR5A1 are known to cause 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency, such mutations have not been implicated in testicular development of 46,XX gonads. Here, we identified identical NR5A1 mutations in two unrelated Japanese patients with 46,XX testicular/ovotesticular DSD. The p.Arg92Trp mutation was absent from the clinically normal mothers and from 200 unaffected Japanese individuals. In silico analyses scored p.Arg92Trp as probably pathogenic. In vitro assays demonstrated that compared with wild‐type NR5A1, the mutant protein was less sensitive to NR0B1‐induced suppression on the SOX9 enhancer element. Other sequence variants found in the patients were unlikely to be associated with the phenotype. The results raise the possibility that specific mutations in NR5A1 underlie testicular development in genetic females.     

Biochemical characterization of a shikimic acid-resistant mutant of Nostoc linckia

The growth of Nostoc linckia was significantly inhibited by shikimic acid concentrations greater than 4.0 μg ml^?1 and was completely inhibited at 10.0μg ml^?1. Shikimic acid increased the duration of the lag phase and the doubling time and hastened the onset of the retardation phase of growth. A mutant (NL_shi) capable of growing in presence of 50 μg ml^?1 shikimic acid, was isolated by nitrosoguanidine mutagenesis from the wild type population at a frequency of about 1 × 10^?5 to 1 × 10^?6. The mutant grew slower than the wild type. Both the wild type and the mutant strain grew photoheterotrophically in light, with and without 3 (3–4 dichlorophenyl) 1,1-dimethyl urea (DCMU) and in darkness when provided with glucose. Glucose supplementation promoted ammonium uptake from the medium, when wild type and mutant were grown in an ammonium-supplemented medium. Glucose stimulated heterocyst production and nitrogenase activity in both the strains. As compared to wild type, this mutant showed higher heterocyst frequency and nitrate reductase activity but its ammonium uptake activity was lower. No significant difference in glutamine synthetase and nitrogenase activities of the mutant were observed. The mutant was stable and retained its resistance even after several subcultures through medium free of shikimic acid.     

<Emphasis Type="Italic">RNR4</Emphasis> mutant alleles <Emphasis Type="Italic">pso3-1</Emphasis> and <Emphasis Type="Italic">rnr4Δ</Emphasis> block induced mutation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Δ mutant that allows only very little mutation induction at very low doses of 254_nm ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Δ mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G → A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant’s protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Δ mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele.     

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K⁺ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (I _to) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (I _K1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca²⁺] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected I _to and I _K1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in I _K1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with I _K1.     

Subunit assembly in vivo of Escherichia coli RNA polymerase: role of the amino-terminal assembly domain of alpha subunit

Background: The RNA polymerase core enzyme of Escherichia coli is assembled in the sequence α→α₂→α₂β→α₂ ββ′. The amino-terminal domain down to residue 235 of the Escherichia coli RNA polymerase α subunit plays a key role in enzyme assembly. In vitro reconstitution studies from mutant α subunits have indicated the involvement of multiple sites for α dimerization, two regions (one near residue 45 and the other near residue 80) for β association, and two regions (one around residue 80 and the other between residues 180 and 200) for β′ association. The mechanism of RNA polymerase assembly in vivo, however, remains largely unknown. Results: RNA polymerase assembly in vivo was analysed for E. coli strains carrying expression plasmids for four amino-terminal deletion and 11 Ala-Ser (AS) dipeptide-insertion mutant α subunits. For detection of RNA polymerase complexes, a hexa-histidine (H₆) tag was added to all these mutant α at their carboxy-termini, and subunit complexes containing the H₆-tagged α were isolated by passing cell extracts through Ni²⁺-affinity columns. The assembly properties of most α mutants were consistent with those observed in in vitro reconstitution studies. Some mutants defective in β′ association in vitro such as those carrying mutations at residues 80 and 200 were, however, assembled in vivo, suggesting that a specific condition(s) or factor(s) supports RNA polymerase assembly in vivo. One possible candidate supporting the RNA polymerase assembly is the molecular chaperon(s), because DnaK (hsp70) was always associated with assembly-defective RNA polymerase mutants. Most assembly competent mutants complemented two temperature-sensitive mutant alleles of rpoA, but two assembly competent mutants, one (αΔN20) carrying a deletion at the extreme amino-terminal region and the other (αI-60) with AS insertion at residue 60, failed to complement these ts mutants. The failure suggests that these assembly competent but complementation-negative α mutants lack an as yet unidentified function(s). In the case of these two mutants, DnaK was associated, with apparently assembled RNA polymerase. Conclusion: The α–α, α–β and α–β′ contact sites on the RNA polymerase α subunit identified in in vitro reconstitution studies also participate in the subunit assembly in vivo. Some α mutants defective in assembly in vitro are, however, assembled in vivo. A factor(s) such as the molecular chaperon DnaK or a specific intracellular condition(s) may affect RNA polymerase assembly in vivo.     

bis-Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs

Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.     

High hydrostatic pressure-induced inactivation of bacterial spores

Abstract

Mycobacterium tuberculosis (Mtb) and other members of the Mtb complex possess an expanded complement of genes for the biosynthesis of molybdenum cofactor (MoCo), a tricyclic pterin molecule that is covalently attached to molybdate. This cofactor allows the redox properties of molybdenum to be harnessed by enzymes in order to catalyze redox reactions in carbon, nitrogen and sulfur metabolism. In this article, we summarize recent advances in elucidating the MoCo biosynthetic pathway in Mtb and highlight the evidence implicating the biosynthesis of this cofactor, as well as the enzymes that depend upon it for activity, in Mtb pathogenesis.     

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2

Numerous studies have indicated that nano-titanium dioxide (TiO₂) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO₂ affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO₂ for 24 h, and nano-TiO₂ internalization, dendritic growth, glutamate metabolism, expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (I_Na) and potassium current (I_K) were examined. Our findings demonstrated that nano-TiO₂ crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO₂ induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca²⁺]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the I_Na and I_K. In addition, nano-TiO₂ increased nitric oxide and nitrice synthase, attenuated the activities of Ca²⁺-ATPase and Na⁺/K⁺-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO₂ inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function.