Involvement of chlA, E, M, and N loci in Escherichia coli molybdopterin biosynthesis.

All molybdenum enzymes except nitrogenase contain a common molybdenum cofactor, whose organic moiety is a novel pterin called molybdopterin (MPT). To assist in elucidating the biosynthetic pathway of MPT, two MPT-deficient mutants of Escherichia coli K-12 were isolated. They lacked activities of the molybdenum enzymes nitrate reductase and formate dehydrogenase, did not reconstitute apo nitrate reductase from a Neurospora crassa nit-1 strain, and did not yield form A, a derivative of MPT. By P1 mapping, these two mutations mapped to chlA and chlE, loci previously postulated but never definitely shown to be involved in MPT biosynthesis. The two new mutations are in different genetic complementation groups from previously isolated chlA and chlE mutations and have been designated as chlM and chlN (closely linked to chlA and chlE, respectively). The reported presence of Mo cofactor activity in the chlA1 strain is shown to be due to in vitro synthesis of MPT through complementation between a trypsin-sensitive macromolecule from the chlA1 strain and a low-molecular-weight compound from the nit-l strain.     

Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli.

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.     

Involvement of a protein with molybdenum cofactor in the in vitro activation of nitrate reductase from a chlA mutant of Escherichia coli K12

Chlorate-resistant mutants are pleiotropically defective in molybdoenzyme activities. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4), which is present in cell-free extracts of chlA mutants can be activated by addition of purified protein PA, the presumed active product of the chlA+ locus, but the activity of the purified protein PA is low, since comparatively large amounts of protein PA are required for the activation. Addition of 10 mM tungstate to the growth medium of a chlBchlC double mutant leads to inactivation of both the molybdenum cofactor and protein PA. Protein PA prepared from such cells was unable to potentiate the in vitro activation of nitrate reductase present in the soluble fraction of a chlA mutant. Quantitation of inactive protein PA was determined immunologically using protein PA-specific antiserum. When a heat-treated extract of a wild-type strain was added to purified protein PA or to the supernatant fraction of a chlBchlC double mutant grown with tungstate, a large stimulation in the ability of these preparations to activate chlA nitrate reductase was found. We equate the activator of protein PA with molybdenum cofactor because: (1) both are absent from heated extracts of tungstate-grown chlBchlC double mutant and cofactor defective chlA and chlE mutants; (2) both are present in heated extracts of wild-type strain; and (3) they behave identically on molecular-sieve columns.     

In vitro system for molybdopterin biosynthesis.

A high-Mr fraction present in chl+ and chlA1 strains of Escherichia coli synthesizes molybdopterin (MPT) from the low-Mr fraction of several MPT-deficient mutants. Using this in vitro complementation as an assay, we have partially characterized the high-Mr fraction as a protein, termed MPT converting factor, of Mr 45,000, distinguishable from the Mo cofactor carrier protein of similar Mr by its absolute requirement for the low-Mr fraction of a non-chlA1 mutant in the nit-1 reconstitution assay. MPT converting factor was rapidly inactivated in the absence of a reduced sulfhydryl compound. Anaerobic incubation of MPT converting factor with trypsin destroyed its activity. High-performance liquid chromatographic analysis of alkaline KMnO4 oxidation products demonstrated that the factor did not contain any bound pterin. Since mutants lacking MPT converting factor are not auxotrophs for folate or riboflavin, the factor appears to be distinct from known pteridine biosynthetic enzymes in E. coli. We have partially purified and characterized the low-Mr fractions as probable MPT precursors. Several distinct precursors were separable by high-performance liquid chromatography. Like MPT activity, precursor activity was oxygen sensitive. Precursor activity was not correlated with levels of L-threo-neopterin, a major pterin of unknown function in E. coli. Precursor activity was correlated with levels of a new 6-alkylpterin, compound Z, produced by acidic iodine oxidation. Compound Z has the properties expected of an oxidized MPT precursor.     

In vitro restoration of nitrate reductase: investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants.

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10-minus 3 M Na2 MoO4 was active in the restoration assay. Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxE-14, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract. The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 mu g molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.     

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.     

Different forms of molybdenum cofactor inVicia faba seeds: The presence of molybdenum cofactor carrier protein and its purification

There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a low-molecular-mass form (free Mo-co) and in a protein-bound form, in seeds of sevenVicia faba genotypes. Low-molecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from theNeurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of theN. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.Abbreviations CP carrier protein - Mo-co molybdenum cofactor - NR nitrate reductase - XO xanthine oxidase     

Molecular characterization of mutations of nlt-4, the pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa

The nit-4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit-4 mutants, alleles 15, 1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit-4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild-type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine-rich domain which is essential for nit-4 function.     

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.     

Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate. Involvement of molybdenum cofactor

The chlorate-resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell-free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus. Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain. The inclusion of a heat-treated preparation of a wild-type or chlB strain prepared after growth in the absence of tungstate, restores the protein-FA-dependent activation of nitrate reductase. All attempts to activate nitrate reductase in extracts prepared from tungstate-grown wild-type Escherichia coli strains failed. It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate-grown chlB mutant. Heat-treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation. Fractionation by gel filtration of the heat-treated preparation from a wild-type strain produced two active peaks in the eluate of approximate Mr 12000 and less than or equal to 1500. The active material in the heat-treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr less than or equal to 1500. The active material is therefore of low molecular mass and can exist either in a protein-bound form or in an apparently free state. Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit-1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate-grown chlB mutant soluble fraction. This was the case even after proteinase treatment of the heat-stable fraction. Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component. Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non-heat-treated soluble subcellular fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molybdenum cofactor in chlorate-resistant and nitrate reductase-deficient insertion mutants of Escherichia coli

We examined molybdenum cofactor activity in chlorate-resistant (chl) and nitrate reductase-deficient (nar) insertion mutants and wild-type strains of Escherichia coli K-12. The bacterial molybdenum cofactor was assayed by its ability to restore activity to the cofactor-deficient nitrate reductase found in the nit-1 strain of Neurospora crassa. In the wild-type E. coli strains, molybdenum cofactor was synthesized constitutively and found in both cytoplasmic and membrane fractions. Cofactor was found in two forms: the demolybdo form required additional molybdate in the assay mix for detection, whereas the molybdenum-containing form was active without additional molybdate. The chlA and chlE mutants had no detectable cofactor. The chlB and the narG, narI, narK, and narL (previously designated chlC) strains had cofactor levels similar to those of the wild-type strains, except the chlB strains had two to threefold more membrane-bound cofactor. Cofactor levels in the chlD and chlG strains were sensitive to molybdate. When grown in 1 microM molybdate, the chlD strains had only 15 to 20% of the wild-type levels of the demolybdo and molybdenum-containing forms of the cofactor. In contrast, the chlG strains had near wild-type levels of demolybdo cofactor when grown in 1 microM molybdate, but none of the molybdenum-containing form of the cofactor. Near wild-type levels of both forms of the cofactor were restored to the chlD and chlG strains by growth in 1 mM molybdate.     

Molybdoenzyme biosynthesis in Escherichia coli: in vitro activation of purified nitrate reductase from a chlB mutant.

All molybdoenzyme activities are absent in chlB mutants because of their inability to synthesize molybdopterin guanine dinucleotide, which together with molybdate constitutes the molybdenum cofactor in Escherichia coli. The chlB mutants are able to synthesize molybdopterin. We have previously shown that the inactive nitrate reductase present in a chlB mutant can be activated in a process requiring protein FA and a heat-stable low-molecular-weight substance. We show here that purified nitrate reductase from the soluble fraction of a chlB mutant can be partially activated in a process that requires protein FA, GTP, and an additional protein termed factor X. It appears that the molybdopterin present in the nitrate reductase of a chlB mutant is converted to molybdopterin guanine dinucleotide during activation. The activation is absolutely dependent upon both protein FA and factor X. Factor X activity is present in chlA, chlB, chlE, and chlG mutants.     

Reconstitution of Nitrate Reductase Activity and Formation of Membrane Particles from Cytoplasmic Extracts of Chlorate-Resistant Mutants of Escherichia coli

The reconstitution of nitrate reductase activity in mixtures of cytoplasmic fractions from the chlorate-resistant mutants chlA, B, C, and E which are lacking this activity was investigated, and the membrane-like particulate material which formed during this reconstitution was analyzed by polyacrylamide gel electrophoresis. When chlA and chlB extracts are incubated together, the cytoplasmic membrane proteins present in the particles which are formed are contributed by both mutants, and the proteins are essentially the same as the proteins in the cytoplasmic membrane fractions of the two mutants. Identical amounts of protein become particulate when cytoplasmic extracts of any of the mutant strains or wild-type strains are incubated at 32 C either singly or in mixtures, and the formation of particulate material does not appear to be a consequence of nitrate reductase reconstitution. Experiments with wild-type strains indicate that the membrane proteins in the cytoplasmic extract are derived from the cytoplasmic membrane during cell breakage. Reconstitution experiments involving various combinations of preincubated and unincubated extracts of the mutants have allowed a preliminary identification of three types of components which are necessary for the formation of active nitrate reductase: (i) a soluble factor present only in extracts from induced chlB; (ii) a different soluble factor which is missing in chlB but is present in extracts from wild-type, chlA, chlC, and chlE; and (iii) a complex including the nitrate reductase protein which is inactivated by preincubation of the mutant extracts.     

Site-directed mutagenesis of the ''zinc finger'' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.     

In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin

The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.     

Evidence for MoeA-dependent formation of the molybdenum cofactor from molybdate and molybdopterin in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The function of the MoeA protein in the biosynthesis of the molybdenum cofactor (MoCo) was analyzed in vitro, using purified His(6)-MoeA from Escherichia coli, molybdopterin (MPT) isolated from buttermilk xanthine oxidase and molybdate. The formation of MoCo was monitored by the reconstitution of nitrate reductase activity in extracts of the Neurospora crassa nit-1 mutant. Formation of MoCo from MPT and molybdate required MoeA and L-cysteine or glutathione. The reaction proceeded at micromolar molybdate levels and was time- and MoeA concentration-dependent. A physical interaction between MoeA and MPT was demonstrated by HPLC analysis of MoeA-bound MPT.     

Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.     

Induction and Repression of Nitrate Reductase in Neurospora crassa

Synthesis of wild-type Neurospora crassa assimilatory nitrate reductase is induced in the presence of nitrate ions and repressed in the presence of ammonium ions. Effects of several Neurospora mutations on the regulation of this enzyme are shown: (i) the mutants, nit-1 and nit-3, involving separate lesions, lack reduced nicotinamide adenine dinucleotide (NADPH)-nitrate reductase activity and at least one of three other activities associated with the wild-type enzyme. The two mutants do not require the presence of nitrate for induction of their aberrant nitrate reductases and are constitutive for their component nitrate reductase activities in the absence of ammonium ions. (ii) An analog of the wild-type enzyme (similar to the nit-1 enzyme) is formed when wild type is grown in a medium in which molybdenum has been replaced by vanadium or tungsten; the resulting enzyme lacks NADPH-nitrate reductase activity. Unlike nit-1, wild type produced this analog only in the presence of nitrate. Contaminating nitrate does not appear to be responsible for the observed mutants' activities. Nitrate reductase is proposed to be autoregulated. (iii) Mutants (am) lacking NADPH-dependent glutamate dehydrogenase activity partially escape ammonium repression of nitrate reductase. The presence of nitrate is required for the enzyme's induction. (iv) A double mutant, nit-1 am-2, proved to be an ideal test system to study the repressive effects of nitrogen-containing metabolites on the induction of nitrate reductase activity. The double mutant does not require nitrate for induction of nitrate reductase, and synthesis of the enzyme is not repressed by the presence of high concentrations of ammonium ions. It is, however, repressed by the presence of any one of six amino acids. Nitrogen metabolites (other than ammonium) appear to be responsible for the mediation of "ammonium repression."     

A common pathway for the activation of several molybdoenzymes in Escherichia coli K12

Three molybdoenzymes, nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase which form part of different systems, have been studied in a parental strain of Escherichia coli K12. When the organism is grown in the presence of 10 mM tungstate, these three enzymes are present in an inactive form which may be activated in vivo by the addition of 1 mM sodium molybdate. The mixing of soluble fractions from chlA and chlB mutants grown under the appropriate conditions leads to the activation of nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase. The activation of each enzyme is maximal when the mutants are grown under conditions that lead to the induction of that enzyme in the wild-type strain. The employment of purified proteins, the association factor FA and the Protein PA, which are presumed to be the products of the chlA and chlB genes, has shown that these proteins are responsible for the activation of the three enzymes during the complementation process.