Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.     

Archaeal shikimate kinase, a new member of the GHMP-kinase family

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.     

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.     

Unique biosynthesis of dehydroquinic acid?

A search of the genomic sequences of the thermophilic microorganisms Aquifex aeolicus, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii for the first seven enzymes (aroG, B, D, E, K, A, and C ) involved in the shikimic acid biosynthetic pathway reveal two key enzymes are missing. The first enzyme in the pathway, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (aroG) and the second enzyme in the pathway, 5-dehydroquinic acid synthase (aroB) are "missing." The remaining five genes for the shikimate pathway in these organism are present and are similar to the corresponding Escherichia coli genes. The genomic sequences of the thermophiles Pyrococcus abyssi and Thermotoga maritima contain the aroG and aroB genes. Several fungi such as Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pneumocystis carinii f. sp. carinii, and Neurospora crassa contain the gene aroM, a pentafunctional enzyme whose overall activity is equivalent to the combined catalytic activities of proteins expressed by aroB, D, E, K, and A genes. Two of these fungi also lack an aroG gene. A discussion of potential reasons for these missing enzymes is presented.     

proP-mediated proline transport also plays a role in Escherichia coli osmoregulation.

Shikimate kinase II was purified to near homogeneity from an Escherichia coli strain which overproduced the enzyme. The apparent Km of this isoenzyme for shikimate was 200 microM, and for ATP it was 160 microM. The Km for shikimate is approximately 100-fold lower than the Km of shikimate kinase I, suggesting that shikimate kinase II is the isoenzyme normally functioning in aromatic biosynthesis. Shikimate kinase II is dependent on metal ions for activity.     

The in-vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts

Full-length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in-vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N-terminal portion of the deduced amino acid sequence resembles known chloroplast-specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in-vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.     

Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.     

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana ^r -6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana ^r -6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.     

The gene for 2-phosphoglycolate phosphatase (gph) in Escherichia coli is located in the same operon as dam and at least five other diverse genes

Downstream of the dam gene in the Escherichia coli genome the following three genes are located: first rpe, then a gene encoding a 27 kDa protein and finally trpS. Here we present evidence that the 27 kDa protein has 2-phosphoglycolate phosphatase activity, and we name the gene gph. Phosphoglycolate phosphatase is needed in autotrophic organisms performing the Calvin-Benson-Bassham (CBB) reductive pentose-phosphate cycle. E. coli is not capable of autotrophic growth and probably utilizes Gph activity for other function(s) than in the CBB cycle. We found no physiological effect of deleting gph and its function in E. coli remains unclear. The use of fusion plasmids, where lacZ was inserted into gph and trpS, and deletion derivatives of these fusion plasmids, showed that rpe, gph and trpS are all members of the dam-containing operon. A novel promoter was identified in the distal part of the dam gene. The operon, which contains aroK, aroB, urf74.3, dam, rpe, gph, and trpS, can be termed a superoperon, since it consists of (at least) seven apparently unrelated genes which are under complex regulatory control.     

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana ^r -6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana ^r -6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity. Received: 13 May 1997 / Accepted: 14 January 1998     

Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein.

Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.     

Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents. The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded approximately 30% of target proteins to total soluble cell proteins. Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-fold increase for EPSP synthase.     

Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria.

The hemin receptor HemR of Yersinia enterocolitica was identified as a 78 kDa iron regulated outer membrane protein. Cells devoid of the HemR receptor as well as cells mutated in the tonB gene were unable to take up hemin as an iron source. The hemin uptake operon from Y. enterocolitica was cloned in Escherichia coli K12 and was shown to encode four proteins: HemP (6.5 kDa), HemR (78 kDa), HemS (42 kDa) and HemT (27 kDa). When expressed in E.coli hemA aroB, a plasmid carrying genes for HemP and HemR allowed growth on hemin as a porphyrin source. Presence of genes for HemP, HemR and HemS was necessary to allow E.coli hemA aroB cells to use hemin as an iron source. The nucleotide sequence of the hemR gene and its promoter region was determined and the amino acid sequence of the HemR receptor deduced. HemR has a signal peptide of 28 amino acids and a typical TonB box at its amino-terminus. Upstream of the first gene in the operon (hemP), a well conserved Fur box was found which is in accordance with the iron-regulated expression of HemR.     

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Molecular characterization and heterologous expression of quinate dehydrogenase gene from <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> IFO3244

The quinate dehydrogenase (QDH) from Gluconobacter oxydans IFO3244 exhibits high affinity for quinate, suggesting its application in shikimate production. Nucleotide sequence analysis of the qdh gene revealed a full-length of 2475-bp encoding an 824-amino acid protein. The qdh gene has the unusual TTG translation initiation codon. Conserved regions and a signature sequence for the quinoprotein family were observed. Phylogenetic analysis demonstrated relatedness of QDH from G. oxydans to other quinate/shikimate dehydrogenases with the highest similarity (56%) with that of Acinetobacter calcoaceticus ADP1 and lower similarity (36%) with a membrane-bound glucose dehydrogenase of Escherichia coli. The function of the gene coding for QDH was confirmed by heterologous gene expression in pyrroloquinoline quinone-synthesizing Pseudomonas putida HK5.