Lysine Biosynthetic Pathway Research Articles

Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase.

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.

Phylogenetic Spectrum of Fungi That Are Pathogenic to Humans

Recent phylogenetic studies based on ribosomal RNA sequences have confirmed that the organisms traditionally treated as fungi include those that have evolved from several different lines (multiphyletic organisms), as has been suspected. Even organisms causing disease in humans represent at least two evolutional lines. Pythium insidiosum and Prototheca species are both believed to have evolved from one line, while the rest of the pathogens have evolved from another line. P. insidiosum is more closely related to red algae and diatoms than to fungi. Prototheca species, as has been previously postulated, are closer to blue-green algae and plants than to fungi. Pythiosis and protothecosis, however, will still be dealt with by medical mycologists because of the morphological and in vivo staining characteristics of the causative organisms. Molecular genetic studies have revealed that Pneumocystis carinii can best be categorized as a fungus, although questions regarding its fungal status may remain unanswered until additional information becomes available on its life cycle, nuclear division, cell-wall chemistry, nutritional uptake pattern, and lysine biosynthetic pathway as well as the ultrastructural characteristics of its cellular components such as the Golgi complex. The phylogeny of the agents of lobomycosis and rhinosporidiosis, although they are treated as fungi, remains unknown. Although there is no in vitro culture system for Loboa loboi and Rhinosporidium seeberi at present, a molecular approach would allow us to reveal their phylogenetic relationship, and we can hope that such attempts are forthcoming.

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.

Cloning, DNA Sequence, Functional Analysis and Transcriptional Regulation of the Genes Encoding Dipicolinic Acid Synthetase Required for Sporulation in Bacillus subtilis

Dipicolinic acid (DPA) is a small polar molecule that accumulates to high concentrations in bacterial endospores, and is thought to play a role in spore heat resistance, or the maintenance of heat resistance. Previous work has shown that mutations in the spo V F locus of Bacillus subtilis prevent the formation of DPA, and give rise to heat-sensitive spores. Addition of exogenous DPA during spore development led to the restoration of heat resistance. This suggested that the spo V F locus encoded dipicolinic acid synthetase, the enzyme thought to catalyse the single reaction needed to synthesise DPA from dihydroxydipicolinic acid, an intermediate in the lysine biosynthetic pathway. We have now cloned and sequenced the spo V F locus of Bacillus subtilis and show that it comprises two coordinately regulated genes, now designated dpaA and dpaB. Expression of fragments of the dpa operon in Escherichia coli has shown that the two gene products together specify DPA synthetase activity. The promoter of the dpa operon, which lies just upstream of the first gene, has been identified by primer extension analysis. Sequences in this region show strong sequence similarity to several promoters recognized by the σ K form of RNA polymerase. Transcription from this promoter was detected four hours after the onset of sporulation, at about the same time that σ K activity is known to appear. Furthermore, transcription was abolished by mutations in a series of genes that are known to be required for the synthesis of active σ K. These results are in accordance with previous work indicating that DPA synthetase activity was present only during the late stages of sporulation and specifically in the mother cell compartment. Transcription was enhanced by a gerE mutation, indicating that, like the previously described cotA gene, spo V F is negatively regulated by GerE. The mother-cell-specific synthesis of an enzyme responsible for a compound that accumulates to high concentrations in the prespore raises interesting questions about intercellular transport mechanisms.

Identification of a lysA-like gene required for tabtoxin biosynthesis and pathogenicity in Pseudomonas syringae pv. tabaci strain PTBR2.024.

Pseudomonas syringae pv. tabaci strain PTBR2.024 produces tabtoxin and causes wildfire disease on tobacco and green bean. PTBR7.000, a Tn5 mutant of PTBR2.024, does not produce tabtoxin, is nonpathogenic on tobacco, and is prototrophic. A 3-kb fragment from a genomic library of the parent strain PTBR2.024 complemented both mutant phenotypes. This 3-kb fragment contains two open reading frames (ORFs), ORF1 and ORF2, and two truncated ORFs, ORF3 and ORF4. The Tn5 insert in PTBR7.000 was mapped to ORF2, and complementation studies showed that an intact ORF2 was sufficient to restore tabtoxin production and pathogenicity. The deduced amino acid sequences of ORF2 and truncated ORF3 contain significant homology to bacterial lysine biosynthetic enzymes, diaminopimelate decarboxylase, and delta 1-piperidine-2,6-dicarboxylate succinyl transferase, respectively. ORF2, however, is not required for lysine biosynthesis. We designated the sequence corresponding to ORF2 as gene tabA and propose that the product of tabA is an enzyme in the tabtoxin biosynthetic pathway that recognizes a substrate analogue of a compound in the lysine biosynthetic pathway.

Physical and biochemical characterization of the cloned LYS5 gene required for ?-aminoadipate reductase activity in the lysine biosynthetic pathway of Saccharomyces cerevisiae

The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of alpha-aminoadipate reductase in the lysine pathway. The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7. An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S. cerevisiae genomic DNA. The lys5+ transformants exhibited alpha-aminoadipate reductase activity similar to that of wild-type cells. S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb. From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product. These results provide a physical and biochemical characterization of the cloned LYS5 gene. Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric alpha-aminoadipate reductase.

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by l-threonine

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by l-threonine. To investigate the regulation of the C. glutamicum HD enzyme by l-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, hom dr. l-Threonine inhibits the wild-type (wt) enzyme with a K i of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM l-threonine. The hom dr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned hom dr gene is genetically unstable. The cloned hom dr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM l-threonine. Sequence analysis revealed that the hom dr mutation is a single nucleotide (G 1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the l-threonine allosteric response. The hom dr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.

Nucleotide sequence of the LYS2 gene of Saccharomyces cerevisiae: homology to Bacillus brevis tyrocidine synthetase 1

The Saccharomyces cerevisiae L YS2 gene, which encodes α-aminoadipate reductase, an essential enzyme in the yeast lysine biosynthetic pathway, has been sequenced. A large open reading frame (ORF) has been identified which can specify a 1392-amino acid protein with a deduced M r of 155 344. A DNA database search using the translated LYS2 ORF as a probe has revealed significant aa sequence homology to the Bacillus brevis enzyme tyrocidine synthetase 1.

Use of α‐aminoadipate and lysine as sole nitrogen source by Schizosacharomyces pombe and selected pathogenic fungi

alpha-Aminodipate, an intermediate of the lysine biosynthetic pathway of fungi, or lysine when used as the sole nitrogen source in the medium was growth inhibitory and toxic to Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe and pathogenic fungi Candida albicans, Filobasidiella neoformans and Aspergillus fumigatus grew in the medium containing alpha-aminoadipate as the sole nitrogen source. C. albicans, A. fumigatus, and one of the strains of F. neoformans also grew in the medium containing lysine as the sole nitrogen source. When grown in the alpha-aminoadipate medium, only S. pombe accumulated a significant amount of alpha-ketoadipate in the culture supernatant. Also, 14C-alpha-aminoadipate was converted to 14C-alpha-ketoadipate in vivo. In the ammonium sulfate medium, S. pombe cells converted 14C-alpha-aminoadipate to lysine. The levels of glutamate-alpha-ketoadipate transaminase, an enzyme responsible for the conversion of alpha-aminoadipate to alpha-ketoadipate, and alpha-aminoadipate reductase, an enzyme required for the conversion of alpha-aminoadipate to lysine, were similar in S. pombe cells grown in the alpha-aminoadipate or ammonium sulfate medium. However, the level of homoisocitrate dehydrogenase, an enzyme before the alpha-ketoadipate step, was twelvefold lower in S. pombe cells grown in the alpha-aminoadipate medium compared to the level in cells grown in the ammonium sulfate medium. Pathogenic fungi used in this study did not accumulate alpha-ketoadipate and alpha-aminoadipate-delta-semialdehyde when grown in medium containing alpha-aminoadipate and lysine, respectively, as sole nitrogen source. However, only pathogenic fungi used both lysine and alpha-aminoadipate as sole nitrogen source. This unique metabolic property could be useful for the identification of these pathogens.

Nucleotide sequence and organization of the upstream region of the Corynebacterium glutamicum lysA gene

Maximum expression of the Corynebacterium glutamicum lysA gene is dependent upon the presence of a 2.3 kb region immediately 5' of the lysA reading frame. Subcloning and functional analysis of the upstream region implied that this region contained the lysA promoter. Sequence determination of the upstream region revealed a single open reading frame, orfX, in the same orientation as lysA. The orfX coding sequence exhibited all the sequence characteristics of a gene with the potential for a 550-amino-acid polypeptide product. Expression of lysA is coupled to that of orfX via a common promoter located immediately 5' of orfX. The RNA start site has been determined by S1 nuclease mapping. Both the orfX and the lysA gene are expressed as a single 3.0 kb RNA transcript. These data indicate that orfX and lysA are genes within a two-gene operon. Expression of the lysA gene is not subject to regulation by lysine. The orfX gene product was shown not to be directly linked to the lysine biosynthetic pathway, nor is it the enzyme incorporating DAP into the peptidoglycan precursor.

Cloning the dapA dapB cluster of the lysine-secreting bacterium Corynebacterium glutamicum

The genes encoding the two successive enzymes of the lysine biosynthetic pathway, dihydrodipicolinate synthase (dapA) and dihydrodipicolinate reductase (dapB), have been isolated from Corynebacterium glutamicum by heterologous complementation of Escherichia coli mutants. The two genes reside on a single 3.8-kb chromosomal fragment. They were subcloned as non overlapping fragments on an E. coli/C. glutamicum shuttle vector and introduced into C. glutamicum. This resulted in overexpression of both enzyme activities which was irrespective of the orientation of the inserts and comparable to that obtained with the large 3.8-kb fragment. Therefore, both genes are located in close proximity to each other on the C. glutamicum chromosome, but are apparently independently transcribed.

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.

Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiae. Evidence for a regulatory role of gene LYS14.

Several enzymes of the lysine pathway of Saccharomyces cerevisiae were found to respond to an induction mechanism mediated by the product of gene LYS14 in the presence of 2-aminoadipate semialdehyde, an intermediate of this pathway. This novel regulatory mechanism appears independent of the specific repression by lysine and of the general control of amino acid biosynthesis. Genes LYS1, LYS9 and LYS14 have been cloned and their DNAs used to assay the corresponding messenger RNAs. The results suggest that the induction mechanism, as well as the specific and general regulations, operate at the transcriptional level. The synthesis of saccharopine dehydrogenase (glutamate-forming), previously shown to require the unlinked genes LYS9 and LYS14, is also affected by the induction mechanism. The leaky auxotrophic behaviour of lys14 mutants is explained by the low basal level of expression of LYS9, the structural gene of this enzyme, in the absence of induction by 2-aminoadipate semialdehyde.

Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway.

Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C. The pH optimum for the reverse reaction was 9.5. The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively. The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum.

The content of alpha-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into alpha-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of alpha-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing alpha-aminoadipic acid accumulation. The repression of alpha-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of alpha-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts alpha-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.

Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase fromGlycine max

A detailed characterization of the lysine biosynthetic pathway in plants is yet to be completed. It is, however, assumed that the diaminopimelic acid pathway exists in the plant kingdom, as commonly described forEscherichia coli.Modification and refinement of lytic complementation, a technique previously utilized in bacterial systems, facilitated the isolation of a functional Diaminopimelate Dehydrogenase gene from aGlycine max nuclear gene library. The isolated gene codes for the enzyme meso-diaminopimelate dehydrogenase. The coding capacity for the enzyme was originally contained on a 6.6kb fragment in a Charon 4a soybean gene bank. Subcloning of the 6.6kb fragment resulted in the recombinant plasmid pMW75. Subsequent subcloning resulted in a 4.05 kb fragment contained in pLW14. One region of homology was observed upon hybridization to EcoR1 digested soybean DNA. Homologous sequences were also observed in Triticum DNA.Meso-diaminopimelate dehydrogenase activity was demonstrated inGlycine max embryos. Maximum enzymatic activity of the cloned enzyme was observed at a pH of 8.0. The enzyme encoded by the soybean gene has an apparent molecular weight of 67 000.

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation.

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-semialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHDPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of L-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested. In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.

Lysine biosynthesis in the yeast Candida maltosa: properties of some enzymes and regulation of the biosynthetic pathway

AbstractFour enzymes of the lysine biosynthetic pathway of Candida maltosa were investigated and the regulatory pattern was established. α‐Aminodipate reductase (Mr 160,000), saccharopine reductase (Mr 90,000) and saccharopine dehydrogenase (Mr 45,000) were separated from each other. Homocitrate synthase was sensitive to the separation methods used. The formation of saccharopine reductase was constitutive, whereas the synthesis of homocitrate synthase, α‐aminoadipate reductase and saccharopine dehydrogenase were regulated by the general control of amino acid biosynthesis. l‐Lysine (Ki = 5.0 mm), l‐thialysine (Ki = 15 mm) and dl‐α‐aminoadipate (Ki = 5.1 mm) inhibited homocitrate synthase, the first enzyme of the pathway. The inhibition was competitive with respect to 2‐oxoglutarate. The Km‐values of the enzyme were estimated to be 0.033 mm for acetyl‐CoA and 0.025 mm for 2‐oxoglutarate. The Michaelis constants of the α‐aminoadipate reductase were calculated to be 0.20 mm for α‐aminoadipate, 0.50 mm for ATP, 0.10 mm for NADPH and 3.8 mm for MgCl2. The Km‐values of the saccharopine reductase were 0.55 mm for saccharopine and 0.13 mm for NADP+, respectively. The values of the saccharopine dehydrogenase were estimated to be 1.6 mm for l‐lysine, 0.66 mm for 2‐oxoglutarate and 0.23 mm for NADH. Furthermore, a number of amino acids were found to be effectors of the enzyme reactions.

Lysine biosynthesis and the evolution-of pennate marine diatoms

The classification of lysine biosynthetic pathways in various organisms have been used to investigate their descent in evolution. We have attempted these determinations in the diatoms Amphora coffeaeformis var: perpusilla (Grunow Cleve.) and Phaeodactylum tricornutum (Bohlin). Additionally, we have verified earlier results of Vogel in a green alga, Chlorella pyrenoidosa strain Tx 71105 (Texas Culture Collection). Our research indicates that the diaminopimelic acid route is involved in all three organisms. While these studies do not exclude the possible co-existence of the α-aminoadipic acid route, the results imply a closer evolutionary relationship of pennate diatoms to bacteria and “classical” photosynthetic plants rather than to heterotrophic or mixotrophic fungi and atypical algal strains such as the Euglenophyta.

Use oflysC-lacZfusion for the isolation of regulatory mutants in the lysine biosynthetic pathway

Use of<i>lysC-lacZ</i>fusion for the isolation of regulatory mutants in the lysine biosynthetic pathway