Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes

5-Keto-l-idionic acid (5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv `Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

Circadian Rhythm in Amino Acid Uptake by Synechococcus RF-1

In the prokaryote Synechococcus RF-1, circadian changes in the uptake of l-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in l-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a free-running period of about 24 hours in L/L at 29°C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0°C). The free-running periodicity was “temperature-compensated” from 21 to 37°C. A 24 hour depletion of extracellular Ca²⁺ before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO₃. The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L.     

The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.)

The incorporation of the radioactivity from acetate-1-¹⁴C, acetate-2-¹⁴C, dl-methionine-1-¹⁴C, dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, dl-allyl-glycine-2-¹⁴C, and dl-2-amino-5-hydroxyvalerate-2-¹⁴C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, and acetate-2-¹⁴C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-¹⁴C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydrolyase (deaminating) in a species of Corynebacterium

1. Three bacterial isolates capable of growth on l-threonine medium only when supplemented with branched-chain amino acids, and possessing high l-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that l-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of l-threonine 3-dehydrogenase or l-threonine aldolase in threonine catabolism. 3. l-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [S_0.5], was 40mm and the Hill coefficient (h) was 2.0. l-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60μm, but did not affect the Hill constant. At the fixed l-threonine concentration of 10mm, the effect of l-valine was biphasic, progressive activation occurring at concentrations up to 2mm-l-valine, but was abolished by higher concentrations. Substrate-saturation plots for the l-valine-activated enzyme exhibited normal Michaelis–Menten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the `biosynthetic' isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of l-threonine dehydratase was constitutive and was not subject to multivalent repression by l-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of l-threonine, apparently initiated by a `biosynthetic' l-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.     

A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily: PRODUCTION,FUNCTIONAL CHARACTERIZATION,AND STRUCTURE MODELING*

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.     

Overproduction of l-Cysteine and l-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

Organisms that overproduced l-cysteine and l-cystine from glucose were constructed by using Escherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg of l-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production of l-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production of l-cysteine plus l-cystine was markedly increased compared to production in JM39.l-Cysteine, one of the important amino acids used in the pharmaceutical, food, and cosmetics industries, has been obtained by extracting it from acid hydrolysates of the keratinous proteins in human hair and feathers. The first successful microbial process used for industrial production of l-cysteine involved the asymmetric conversion of dl-2-aminothiazoline-4-carboxylic acid, an intermediate compound in the chemical synthesis of dl-cysteine, to l-cysteine by enzymes from a newly isolated bacterium, Pseudomonas thiazoliniphilum (11). Yamada and Kumagai (13) also described enzymatic synthesis of l-cysteine from beta-chloroalanine and sodium sulfide in which Enterobacter cloacae cysteine desulfhydrase (CD) was used. However, high level production of l-cysteine from glucose with microorganisms has not been studied.Biosynthesis of l-cysteine in wild-type strains of Escherichia coli and Salmonella typhimurium is regulated through feedback inhibition by l-cysteine of serine acetyltransferase (SAT), a key enzyme in l-cysteine biosynthesis, and repression of expression of a series of enzymes used for sulfide reduction from sulfate by l-cysteine (4), as shown in Fig. ​Fig.1.1. Denk and Böck reported that a small amount of l-cysteine was excreted by a revertant of a cysteine auxotroph of E. coli. In this revertant, SAT encoded by the cysE gene was desensitized to feedback inhibition by l-cysteine, and the methionine residue at position 256 in SAT was replaced by isoleucine (2). These results indicate that it may be possible to construct organisms that produce high levels of l-cysteine by amplifying an altered cysE gene. Although the residue at position 256 is supposedly part of the allosteric site for cysteine binding, no attention has been given to the effect of an amino acid substitution at position 256 in SAT on feedback inhibition by l-cysteine and production of l-cysteine. It is also not known whether isoleucine is the best residue for desensitization to feedback inhibition. Open in a separate windowFIG. 1Biosynthesis and regulation of l-cysteine in E. coli. Abbreviations: APS, adenosine 5′-phosphosulfate; PAPS, phosphoadenosine 5′-phosphosulfate; Acetyl CoA, acetyl coenzyme A. The open arrow indicates feedback inhibition, and the dotted arrows indicate repression.On the other hand, l-cysteine appears to be degraded by E. coli cells. Therefore, in order to obtain l-cysteine producers, a host strain with a lower level of l-cysteine degradation activity must be isolated. In this paper we describe high-level production of l-cysteine plus l-cystine from glucose by E. coli resulting from construction of altered cysE genes. The methionine residue at position 256 in SAT was replaced by other amino acids or the termination codon in order to truncate the carboxy terminus from amino acid residues 256 to 273 by site-directed mutagenesis. A newly derived cysteine-nondegrading E. coli strain with plasmids having the altered cysE genes was used to investigate production of l-cysteine plus l-cystine.     

Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters,SerP1 and SerP2, of Lactococcus lactis

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports l-serine, l-threonine, and l-cysteine with high affinity. Affinity constants (K_m) are in the 20 to 40 μM range. SerP2 is a dl-alanine/dl-serine/glycine transporter. The preferred substrate appears to be dl-alanine for which the affinities were found to be 38 and 20 μM for the d and l isomers, respectively. The common substrate l-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 μM, respectively. Growth experiments demonstrate that SerP1 is the main l-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates l-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic d-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of d-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.     

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

Purification and characterization of dihydrodipicolinate synthase from pea

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-β-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-β-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-α-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-α-(2-aminoethoxyvinyl)glycine is noncompetitive towards l-aspartate-β-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-β-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.     

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO₄. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H⁺/10⁶ cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-¹⁴C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K⁺ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.     

Commentary on Urinary l-erythro-β-hydroxyasparagine: a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase

The analysis of the urine contents can be informative of physiological homoeostasis, and it has been speculated that the levels of urinary d-serine (d-ser) could inform about neurological and renal disorders. By analysing the levels of urinary d-ser using a d-ser dehydratase (DSD) enzyme, Ito et al. (Biosci. Rep.(2021) 41, BSR20210260) have described abundant levels of l-erythro-β-hydroxyasparagine (l-β-EHAsn), a non-proteogenic amino acid which is also a newly described substrate for DSD. The data presented support the endogenous production l-β-EHAsn, with its concentration significantly correlating with the concentration of creatinine in urine. Taken together, these results could raise speculations that l-β-EHAsn might have unexplored important biological roles. It has been demonstrated that l-β-EHAsn also inhibits serine racemase with K_i values (40 μM) similar to its concentration in urine (50 μM). Given that serine racemase is the enzyme involved in the synthesis of d-ser, and l-β-EHAsn is also a substrate for DSD, further investigations could verify if this amino acid would be involved in the metabolic regulation of pathways involving d-ser.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Metabolic and chemical properties of basic proteins isolated from nuclei of rat liver and thymus gland

1. The effects of alkylating agents and disulphides on the thiol-containing proteins of nuclei from rat thymus and liver were studied. Three protein fractions were examined: histones extracted with 50mm- and 250mm-hydrochloric acid and the residual protein. None of the reagents selectively reacted with any one of the protein fractions. 2. Amino acid uptake in vitro into the histones of nuclei from rat thymus was analysed by preparative electrophoresis of the proteins extracted with 50mm- and 250mm-hydrochloric acid. After 1hr. at 37° the greater incorporation was into the proteins extracted with 50mm-hydrochloric acid. 3. Preparative electrophoresis was used to study the relative thiol contents of the proteins of the 50mm-hydrochloric acid extract from thymus nuclei by labelling the histones in vitro with ¹⁴C-labelled N-ethylmaleimide. 4. The capacity of the proteins extracted from rat thymus with 50mm- and 250mm-hydrochloric acid, and of the components from these extracts separated by preparative electrophoresis, to combine with DNA and to depress DNA-dependent RNA synthesis was studied. The histones extracted with 50mm-hydrochloric acid were more lysine-rich than those extracted with 250mm-hydrochloric acid. Wide variations were found in the abilities of the separated components to depress RNA synthesis.