In Vitro Metabolism of the Stereoisomers of 2,6-Diaminopimelic Acid by Mixed Rumen Protozoa and Bacteria

Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria. Received: 16 March 1996 / Accepted: 14 May 1996     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.     

Molecular Evolution of the Lysine Biosynthetic Pathways

Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes. One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA). A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al. 1999). Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them. No evidence was found of an evolutionary relationship between the DAP and AAA enzymes. Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism. This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al. 2001). Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al. 2001). Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.     

Cloning and Sequencing of the meso-Diaminopimelate-d-dehydrogenase (ddh) Gene of Corynebacterium glutamicum

The ddh gene of Corynebacterium glutamicum encoding raesodiaminopimelate meso-diaminopimelate (meso-DAP)-d-dehydrogenase (DDH) involved in the lysine biosynthesis was cloned in a DAP auxotroph (dapD4) of Escherichia coli by complementation of the DAP auxotroph. Deletion analysis revealed that a ~1.7-kb XhoI-KpnI fragment contained the ddh structure gene. The specific activity of DDH was increased fourteen-fold when C. glutamicum was transformed with a recombinant plasmid harbouring the cloned ddh gene. Furthermore, the ddh gene has been sequenced [S. Ishino et al., Nucleic Acids Res., 15, 3917 (1987)] and some properties of the ddh gene are discussed.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Development of stable, genetically well-defined conditionally viableEscherichia coli strains

Summary The diaminopimelate (DAP) pathway provides the cell with lysine and with DAP, a vital cell wall constituent. Mutations in the DAP pathway of lysine biosynthesis are lethal for cells exposed to lysine in the absence of DAP. In this paper, the substitution of thedapD gene ofEscherichia coli with the kanamycin resistance gene from Tn903 is described and its possible uses are discussed.     

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-Å X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

Isolation of bioactive actinomycetes from marine sediments using rifampicin

Summary Bioactive actinomycetes were isolated from marine sediments using rifampicin. Plating the sediments on starch-casein agar, supplemented with rifampicin, eliminated the occurrence of contaminating microorganisms. Total counts, however, were reduced in the presence of rifampicin. Most of the isolates contained ll-2,6-diaminopimelic acid (DAP), whereas 37% contained meso-DAP. The use of increasing concentrations of rifampicin tended to yield a higher proportion of strains with cell extracts positive for meso-DAP. Streptomyces and Micromonospora represented the major genera identified. Antimicrobial activity was exhibited by 46% of the isolates, primarily against Gram-positive bacteria. Inhibition of Gram-negative bacteria was minimal, but antimycotic activity was displayed by 28% of the actinomycetes. Most of the latter activity was attributable to polyenes, particularly hexanenes. The results obtained indicate that rifampicin, added to starch-casein agar, is effective for the isolation of bioactive actinomycetes from marine sediments.     

Crystal Structure of Diaminopimelate Epimerase from Arabidopsis thaliana, an Amino Acid Racemase Critical for l-Lysine Biosynthesis

Diaminopimelate (DAP) epimerase is a key enzyme for the biosynthesis of lysine in plants. Lysine is an essential dietary nutrient for mammals. In both plants and bacteria, DAP epimerase catalyzes the interconversion of ll-DAP and dl(meso)-DAP. The absence of a mammalian homolog makes DAP epimerase a promising target for the design of novel herbicides and antibacterials. This enzyme requires no cofactors and it functions through an unusual mechanism involving two cysteine residues acting in concert and alternating as a base (thiolate) and as an acid (thiol). The present study reports the crystal structures of two enzyme-inhibitor complexes of DAP epimerase from Arabidopsis thaliana with different isomers of the irreversible inhibitor and substrate mimic, 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 Å resolution. These structures provide the first atomic details of a plant amino acid racemase. Structural analysis reveals that ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with two strictly conserved cysteine residues, Cys99 and Cys254, optimally positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic α-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the l and d stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the l configuration. Structural comparisons of the plant enzyme with its bacterial counterpart from Haemophilus influenzae reveal significant conservation of amino acid residues around the active site that extends to their three-dimensional structures and catalytic mechanism.     

Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis

The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Δmmp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and α-ketoglutarate as substrates was 24.3 ± 2.0 nmol min⁻¹ mg of protein⁻¹. The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70°C and pH 8.0 to 9.0. The apparent K_ms of MJ1391 for ll-DAP and α-ketoglutarate were 82.8 ± 10 μM and 0.42 ± 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales.Two lysine biosynthesis pathways evolved separately in organisms, the diaminopimelic acid (DAP) and aminoadipic acid (AAA) pathways. The DAP pathway synthesizes l-lysine from aspartate and pyruvate, and diaminopimelic acid is an intermediate. This pathway is utilized by most bacteria, some archaea, some fungi, some algae, and plants (28, 29). The AAA pathway synthesizes l-lysine from α-ketoglutarate and acetyl coenzyme A (acetyl-CoA), and α-aminoadipic acid is an intermediate. This pathway is utilized by most fungi, some algae, the bacterium Thermus thermophilus, and probably some archaea, such as Sulfolobus, Thermoproteus, and Pyrococcus (27, 36). No organism is known to possess both pathways.There are four known variations of the DAP pathway in bacteria: the succinylase, acetylase, aminotransferase, and dehydrogenase pathways (Fig. ​(Fig.1).1). These pathways share the steps converting l-aspartate to l-2,3,4,5-tetrahydrodipicolinate (THDPA), but the subsequent steps leading to the production of meso-diaminopimelate, the immediate precursor of l-lysine, are different. The succinylase pathway acylates THDPA with succinyl-CoA to generate N-succinyl-ll-2-amino-6-ketopimelate and forms meso-DAP by subsequent transamination, desuccinylation, and epimerization. This pathway is utilized by proteobacteria and many firmicutes and actinobacteria (12, 14, 20, 29). The acetylase pathway is analogous to the succinylase pathway but uses N-acetyl intermediates. This pathway is limited to certain Bacillus species, in which the corresponding genes have not been identified (33, 39). The aminotransferase pathway converts THDPA directly to ll-DAP by diaminopimelate aminotransferase (DapL) without acylation. This pathway is shared by cyanobacteria (19), chlamydia (24), the archaeon Methanothermobacter thermautotrophicus (15, 18), and the plant Arabidopsis thaliana (19). The dehydrogenase pathway forms meso-DAP directly from THDPA, NADPH, and NH₄⁺ by using diaminopimelate dehydrogenase (Ddh). This pathway is utilized by some Bacillus and Brevibacterium species and Corynebacterium glutamicum (25, 26, 40). Most bacteria use only one of the four variants, although certain bacteria, such as C. glutamicum and Bacillus macerans, possess both the succinylase and dehydrogenase pathways (3, 30).Open in a separate windowFIG. 1.Variations in the DAP pathway for lysine biosynthesis. 1, succinylase pathway; 2, acetylase pathway; 3, aminotransferase pathway; 4, dehydrogenase pathway. Abbreviations and designations: THDPA, l-2,3,4,5-tetrahydrodipicolinate; l,l-DAP, ll-2,6-diaminopimelate; meso-DAP, meso-2,6-diaminopimelate; LysC, aspartate kinase; Asd, aspartate semialdehyde dehydrogenase; DapA, dihydrodipicolinate synthase; DapB, dihydrodipicolinate reductase; DapD, THDPA succinylase; DapC, succinyl-DAP aminotransferase; DapE, succinyl-DAP desuccinylase; DapF, DAP epimerase; LysA, DAP decarboxylase; DapL, ll-DAP aminotransferase; Ddh, DAP dehydrogenase.The diaminopimelate aminotransferase (DapL) catalyzes the transfer of an amino group from l-glutamate to THDPA, forming ll-DAP (19, 24). It uses pyridoxal 5′-phosphate (PLP) as a coenzyme and has constrained substrate specificity. DapL is not closely related to the DapC/ArgD aminotransferase, which functions in the succinylase pathway. Comparative genomic analysis identified dapL homologs in both bacterial and archaeal genomes. Homologs of dapD and dapE have not been found in genomes with dapL homologs, suggesting that transamination of THDPA does not require succinylation in these organisms (18). Phylogenetic analysis also suggested classification of DapL into two groups, DapL1 and DapL2, which share ∼30% amino acid sequence identity (18). These two groups both exhibit DapL activity, and they cannot be differentiated by kinetic properties (18, 37). The distribution of the two groups is not obviously associated with specific prokaryotic lineages (18).Methanogens are strictly anaerobic archaea that obtain all or most of their energy for growth from the production of large quantities of methane. All methanogens belong to the Euryarchaeota and are currently classified in six orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, and Methanocellales (23, 41, 42). Biochemical studies of Methanocaldococcus jannaschii and Methanococcus voltae belonging to Methanococcales, Methanospirillum hungatei belonging to Methanomicrobiales, and Methanothermobacter thermautotrophicus belonging to Methanobacteriales suggested that these organisms derive their l-lysine from a DAP pathway, but the studies did not discriminate among the four DAP pathway variations (2, 9, 10, 32). Genome sequence analysis also suggested a DAP pathway in Methanosarcina mazei belonging to Methanosarcinales (8). Recent studies identified a dapL homolog belonging to the DapL1 group in M. thermautotrophicus. The gene product complemented an Escherichia coli dapD dapE double mutant and catalyzed the transamination of DAP to THDPA, suggesting that Methanobacteriales use the DapL pathway for l-lysine biosynthesis (15, 18). Homologs of asd, dapA, dapB, dapF, and lysA have been identified in the genomes of M. maripaludis and M. jannaschii belonging to the Methanococcales, but homologs responsible for the conversion of THDPA to ll-DAP have not been annotated (4, 17). Here we identified methanococcal DapL homologs and demonstrated that the DapL pathway is present in Methanococcales.     

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-eemialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHPA-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.Abbreviations l-ASA l-aspartic-2-semialdehyde - DAP diaminopimelic acid - DPA dipicolinic acid - DHDPA dihydrodipicolinate - AGM aspargine-glycerol medium - PY peptone-yeast extract - NB+NSM nutrient broth plus nutrient sporulation medium     

Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum

l-lysine is an essential amino acid that is widely used as a food supplement for humans and animals. meso-Diaminopimelic acid decarboxylase (DAPDC) catalyzes the final step in the de novol-lysine biosynthetic pathway by converting meso-diaminopimelic acid (meso-DAP) into l-lysine by decarboxylation reaction. To elucidate its molecular mechanisms, we determined the crystal structure of DAPDC from Corynebacterium glutamicum (CgDAPDC). The PLP cofactor is bound at the center of the barrel domain and forms a Schiff base with the catalytic Lys75 residue. We also determined the CgDAPDC structure in complex with both pyridoxal 5′-phosphate (PLP) and the l-lysine product and revealed that the protein has an optimal substrate binding pocket to accommodate meso-DAP as a substrate. Structural comparison of CgDAPDC with other amino acid decarboxylases with different substrate specificities revealed that the position of the α15 helix in CgDAPDC and the residues located on the helix are crucial for determining the substrate specificities of the amino acid decarboxylases.     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.     

Regulatory mechanisms after short‐ and long‐term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana

The aspartate‐derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4‐hydroxy‐tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N‐dimethylglycinatoboranyloxycarbonylmethyl)‐dimethylamine‐borane (DDAB) and compared the short‐term effects on the biochemical and biomolecular level to the long‐term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine‐sensitive AKs, AKLYS1 and AKLYS3 control the short‐ and long‐term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.     

A Total Synthesis of (±)-Gibberellin A12 via Methyl (±)-7,16-dioxo-17-norkauran-19-oate

¹⁴C-labelled methionine, xanthosine, and 7-methylxan-thosine were given to excised tea shoots. The methyl group of methionine was incorporated into 7-methylxanthosine (ca. 10%) in the earlier period of incubation after the uptake. About 50% of the radioactivity of xanthosine was rapidly incorporated into caffeine via 7-methylxanthosine, 7-methylxanthine, and theobromine within 24 hr. 7-Methylxanthosine was also converted into caffeine at a high rate. The results suggest that the pathway for caffeine biosynthesis is as follows: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.     

Occurrence of diaminopimelic epimerase in maize

Extracts of maize leaves catalyzed the interconversion of meso-diaminopimelic acid its L-isomer. Three observations support the existence of this epimerase activity: (i) detection of the reversible interconversion of L-diaminopimelic acid and meso-diaminopimelic acid by paper chromatography after incubation of either isomer with extract; (ii) formation of [¹⁴C]CO₂ from L-[¹⁴C]diaminopimelic acid in an incubation mix containing meso-diaminopimelic acid decarboxylase; and (iii) inhibition of [¹⁴C]CO₂ evolution from L-diaminopimelic acid by unlabeled meso-diaminopimelic acid. The demonstration of the diaminopimelic acid epimerase lends support to the occurrence in plants of the complete diaminopimelic acid pathway for biosynthesis of lysine as it occurs in Escherichia coli and most bacteria.     

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.     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.