Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase

Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., L?ufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.     

Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.     

Involvement of quinolinate phosphoribosyl transferase in promotion of potato growth by a Burkholderia strain

Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. In order to determine the mechanism by which this growth stimulation occurs, we used Tn5 mutagenesis to produce a mutant, H41, which exhibited no growth-promoting activity but was able to colonize potato plants as well as the wild-type strain. The gene associated with the loss of growth promotion in H41 was shown to exhibit 65% identity at the amino acid level to the nadC gene encoding quinolinate phosphoribosyltransferase (QAPRTase) in Ralstonia solanacearum. Complementation of H41 with QAPRTase restored growth promotion of potato explants by this mutant. Expression of the gene identified in Escherichia coli yielded a protein with QAPRTase activities that catalyzed the de novo formation of nicotinic acid mononucleotide (NaMN). Two other genes involved in the same enzymatic pathway, nadA and nadB, were physically linked to nadC. The nadA gene was cotranscribed with nadC as an operon in wild-type strain PsJN, while the nadB gene was located downstream of the nadA-nadC operon. Growth promotion by H41 was fully restored by addition of NaMN to the tissue culture medium. These data suggested that QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN.     

Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.     

Recognition of a Gene Involved in the Regulation of Nicotinamide Adenine Dinucleotide Biosynthesis

Mutations affecting the biosynthesis of quinolinic acid, a precursor of nicotinamide adenine dinucleotide (NAD) in Escherichia coli K-12, are either near min 17 (nadA mutants) or near min 49 on the chromosome. These nad mutants all exhibit a phenotypic requirement for NAD or one of its immediate precursors. The mutants with lesions near min 49 can be separated into two groups based on in vitro complementation analysis. One group (nadB) exhibits complementation with nadA mutants, whereas the other group fails to do so. The latter group is tentatively designated nadR based on its regulation of the unlinked nadA gene. The nadR gene maps adjacent to nadB between purI and tyrA.     

The ferrochelatase from Saccharomyces cerevisiae. Sequence, disruption, and expression of its structural gene HEM15

The HEM15 gene in Saccharomyces cerevisiae encodes ferrochelatase (EC 4.99.1.1, protoheme ferrolyase), a mitochondrial inner membrane-bound enzyme which catalyzes the insertion of ferrous ion into protoporphyrin IX, the last step in protoheme biosynthesis. The gene was isolated by functional complementation of a hem15 mutant. Sequence analysis of a 2.9-kilobase genomic DNA fragment revealed an open reading frame of 1179 nucleotides, plus a gene coding for a tRNA(Val)(GUU) and delta elements downstream from the 3'-end of HEM15. The open reading frame encodes a precursor form of the protein containing a 31-amino acid presequence. The mature enzyme contains 362 amino acid residues; its calculated molecular weight (40,900) and predicted amino-terminal sequence agree with those determined from the purified protein. It is relatively abundant in lysine (9%) and contains no apparent transmembrane segment. Disruption of the HEM15 gene led to non-viable cells in certain genetic background. Northern (RNA) analysis showed a slight (1.5-2-fold) repression of HEM15 expression by glucose.     

Cross-Feeding of Escherichia coli Mutants Defective in the Biosynthesis of Nicotinamide Adenine Dinucleotide

Mutants of Escherichia coli defective in the biosynthesis of nicotinamide adenine dinucleotide (NAD) are able to grow in a Casamino Acids medium lacking NAD and its immediate precursors, nicotinic acid and nicotinamide. This property has allowed the development of a system to measure cross-feeding between a nadA and a nadB mutant. This system provides a means of isolating the intermediate, prequinolinic acid, as well as a biological assay for the compound. The nadB mutant feeds the nadA mutant, indicating that the nadA enzyme occurs first in the pathway and the nadB enzyme second. No cross-feeding was detected between nadA and nadC or between nadB and nadC.     

Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints.

We have cloned the Schizosaccharomyces pombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication. The gene was cloned by complementation of the sensitivity to UV light and gamma rays of the rad3-136 mutant with an Sz. pombe genomic library. Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M(r) of 121974. The rad3 gene is a new gene with no homologs in existing sequence databases. The gene is poorly expressed, with a codon bias index of -0.01. A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original rad3-136 mutant. The rad3 gene was mapped to NotI fragment C on chromosome II.     

Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase

Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with l-aspartate oxidase, the other component of the so-called "quinolinate synthetase complex," the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken. The results are presented in this paper. Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up. The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E. coli culture. A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented. Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from l-aspartate, dihydroxyacetone phosphate, and O(2) by using only nadA and nadB gene overexpressed products.     

Genetic characterization and regulation of the nadB locus of Salmonella typhimurium.

The nadB locus encodes the first enzyme of NAD synthesis. It has been reported that this gene and nadA are regulated by a positive regulatory protein encoded in the nadB region. In pursuing this regulatory mechanism, we constructed a fine-structure genetic map of the nadB gene. The region appears to include a single complementation group; no evidence for a positive regulatory element was found. Several mutations causing resistance to the analog 6-aminonicotinamide mapped within the structural gene and probably cause resistance to feedback inhibition. Regulatory mutations for nadB were isolated. These mutants mapped far from nadB near the pnuA gene, which encodes a function required for nicotinamide mononucleotide transport. The regulatory mutations appear to affect a distinct function encoded in the same operon as pnuA.     

The histidine permease gene (HIP1) of Saccharomyces cerevisiae

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.     

Characterization of the Superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg

Abstract A gene ( sod ) encoding Superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identity was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod , separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium .     

Early steps in the biosynthesis of NAD in Arabidopsis start with aspartate and occur in the plastid

NAD is a ubiquitous coenzyme involved in oxidation-reduction reactions and is synthesized by way of quinolinate. Animals and some bacteria synthesize quinolinate from tryptophan, whereas other bacteria synthesize quinolinate from aspartate (Asp) using L-Asp oxidase and quinolinate synthase. We show here that Arabidopsis (Arabidopsis thaliana) uses the Asp-to-quinolinate pathway. The Arabidopsis L-Asp oxidase or quinolinate synthase gene complemented the Escherichia coli mutant defective in the corresponding gene, and T-DNA-based disruption of either of these genes, as well as of the gene coding for the enzyme quinolinate phosphoribosyltransferase, was embryo lethal. An analysis of functional green fluorescent protein-fused constructs and in vitro assays of uptake into isolated chloroplasts demonstrated that these three enzymes are located in the plastid.     

Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I.

As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase. Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene. Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected. We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity. Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases. Wild-type phenotype, however, is only restored with the whole operon. Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors. Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins.     

番茄交替氧化酶基因的克隆和表达

利用简并PCR扩增产物做探针筛选番茄cDNA基因文库获得一个全长交替氧化酶cDNA基因LeAoxlau.经序列分析得出,该基因全长1 418bp,编码区序列长1 077 bp,编码约40 kD的前体蛋白.该蛋白在转运到线粒体时被加工成32kD的成熟蛋白.Southern印迹杂交分析结果显示该基因以单拷贝形式存在于番茄的基因组中RT-PCR显示,该基因在在番茄植株的根、茎、叶和子叶中表达.重组表达实验表明该基因能在大肠杆菌中表达.     

Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes

Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.