On the molecular basis of the high affinity binding of basic amino acids to LAOBP,a periplasmic binding protein from Salmonella typhimurium

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high‐affinity binding of ligands to proteins is still limited; such is the case for l ‐lysine–l ‐arginine–l ‐ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l ‐arginine, l ‐lysine, and l ‐ornithine with nanomolar affinity and to l ‐histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l ‐histidine and l ‐arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~?300 cal mol^?1 K^?1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000‐fold higher affinity of LAOBP for l ‐arginine as compared with l ‐histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy‐driven micromolar affinity toward l ‐arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.     

Arginine and nitrogen mobilization in cyanobacteria

Cyanobacteria have evolved mechanisms to adapt to environmental stress and nutrient availability, including accumulation of storage compounds in inclusions and granules. As arginine is a key building block of cyanophycin, a dynamic nitrogen reservoir in many cyanobacteria, arginine metabolism plays a key role in cyanobacterial nitrogen storage and remobilization. Recently, an arginine dihydrolase AgrE/ArgZ was identified as a major arginine‐degrading enzyme in nondiazotrophic Synechocystis, which catalyzes the conversion of arginine into ornithine and ammonia. The N‐terminal domain of AgrE/ArgZ is responsible for arginine dihydrolase activity. Burnat et al. (2019) identified the arginine catabolic pathway in diazotrophic Anabaena, which starts with the reaction catalyzed by AgrE/ArgZ. Moreover, this study identified the C‐terminal domain of AgrE/ArgZ as an ornithine cyclodeaminase that catalyze the conversion of ornithine to proline. The results demonstrated that arginine is catabolized to generate glutamate by the concerted action of AgrE/ArgZ and bifunctional proline oxidase PutA in the vegetative cells of Anabaena. These findings expand our knowledge on nitrogen mobilization and redistribution in Anabaena under nitrogen‐fixation conditions. AgrE/ArgZ is widely present in many diazotrophic cyanobacteria and may be important for their contribution to marine nitrogen fixation. AgrE/ArgZ may have potential applications in metabolic engineering and biotechnology.     

Selection and characterisation of mutants lacking arginase in Neurospora crassa

Summary A Neurospora mutant (aga) lacking arginase was selected by virtue of its inability to utilize arginine as a source of ornithine, using a strain in which ornithine was needed to satisfy a proline requirement. It mapped in linkage group VII (right arm), close to wc. The most important characteristic of the mutant was its extreme sensitivity to arginine. Inclusion of 1 mM arginine in the medium lead to a 40-fold increase in the arginine pool and a 90% inhibition of growth. This inhibition was relieved by the addition of ornithine or proline. The high arginine pool was associated with only a slight repression of two biosynthetic enzymes examined and with a five-fold induction of ornthine transaminase, the second enzyme of arginine catabolism. It is expected that the aga mutant will be of value in further work on the regulation of arginine biosynthesis in Neurospora.     

The influence of methyl jasmonate,cholesterol and l‐arginine on solasodine production in hairy root culture of Solanum mammosum

The increasing demand of diosgenin for high‐revenue synthesis of steroid hormones by the pharmaceutical industries has driven researchers to look for other alternatives. Solasodine which was reported to be present in Solanum mammosum is known to be a potential source. The present study highlighted that added methyl jasmonate, cholesterol and l ‐arginine into the modified liquid full‐strength Murashige and Skoog (MS) medium (with ammonium to nitrate ratio 10.3 mM: 39.4 mM, and 4% (w/v) sucrose) could influence the solasodine production in the hairy roots of S. mammosum. The findings showed that both hairy root line‐ATCC31798 and line‐A4 (which were separately induced by Agrobacterium rhizogenes strain ATCC31798 and A4) acquired solasodine productivity of 4.5 mg/g dry weight roots with average dry biomass of 190 mg after 32 days culture, when using 50 mg fresh weight roots as initial inoculum size, with 100 mM cholesterol, 1000 μM l ‐arginine and 300 μM methyl jasmonate added simultaneously into the culture medium on day 20 of culture. The amount of solasodine obtained was five times higher than those without both the elicitor and precursor treatment. The improved solasodine production with a high‐biomass growth could reduce the production cost of steroid synthesis in the long run.     

Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture

The characteristics of glucose and amino acid metabolism over a 98-hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2±0.2 to 1.5±0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06±0.01 to 0.82±0.04 mM between 0 and 48 hours and decreased to 0.72±0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD⁺ ratio, as represented by the intracellular lactate/pyruvate ratio (r=0.78–0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70±0.01 to 0.30±0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate-aspartate shuttle, to the cell NADH/NAD⁺ redox state. (3) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production.     

Membrane stabilization by abscisic acid under cold aids proline in alleviating chilling injury in maize (Zea mays L.) cultured cells

Previous studies of maize suspension‐cultured cells showed that abscisic acid (ABA) treatment at warm temperatures improved the tolerance of cells to subsequent chilling. In the present study, it is shown that both ABA‐treated and untreated maize cells accumulated proline in response to chilling. However, ABA‐treated cells displayed less lipid peroxidation during chilling, and thus, unlike untreated cells, were able to retain the accumulated proline intracellularly. Proline application experiments indicate that an intracellular proline level higher than 2 µmole (g FW)^?1 prior to chilling was needed to meaningfully reduce chilling‐enhanced lipid peroxidation and significantly improve chilling tolerance. The results suggest that total proline accumulation in ABA‐treated as well as untreated cells during chilling was enough to potentially improve chilling tolerance, but proline leakage rendered the control cells unable to benefit from the endogenous synthesis of proline in relation to the alleviation of chilling injury. Proline participated in chilling tolerance improvement in ABA‐treated maize cells, as evidenced by: (1) the inhibition of proline accumulation by l ‐methionine‐d , l ‐sulphoximine (MSO), an inhibitor of glutamine synthetase, reduced ABA‐improved chilling tolerance, and (2) the addition of glutamine into the medium prevented the MSO‐induced reduction in chilling tolerance. The revised relationship between proline accumulation and membrane stability at cold is discussed in the light of these current findings.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.     

Propolis reduces oxidative stress in l‐NAME‐induced hypertension rats

The inhibition in the synthesis or bioavailability of nitric oxide (NO) has an important role in progress of hypertension. The blocking of nitric oxide synthase activity may cause vasoconstriction with the formation of reactive oxygen species (ROS). Propolis is a resinous substance collected by honey bees from various plants. Propolis has biological and pharmacological properties. The aim of this study was to examine the effect of propolis on catalase (CAT) activity, malondialdehyde (MDA) and NO levels in the testis tissues of hypertensive rats by Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME). Rats have received nitric oxide synthase inhibitor (l ‐NAME, 40 mg kg^?1, intraperitoneally) for 15 days to produce hypertension and propolis (200 mg kg^?1, by gavage) during the last 5 days. MDA level in l ‐NAME‐treated group significantly increased compared with control group (P < 0.01). MDA level of l ‐NAME + propolis‐treated rats significantly reduced (P < 0.01) compared with l ‐NAME‐treated group. CAT activity and NO level significantly reduced (P < 0.01) in l ‐NAME group compared with control group. There were no statistically significant increases in the CAT activity and NO level of the l ‐NAME + propolis group compared with the l ‐NAME‐treated group (P > 0.01). These results suggest that propolis changes CAT activity, NO and MDA levels in testis of l ‐NAME‐treated animals, and so it may modulate the antioxidant system. Copyright © 2013 John Wiley & Sons, Ltd.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.     

Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Arginine catabolism in Agrobacterium strains: role of the Ti plasmid.

We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium.     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

Arginine and ornithine kinetics in severely burned patients: increased rate of arginine disposal

Arginine serves multiple roles in the pathophysiological response to burn injury. Our previous studies in burn patients demonstrated a limited net rate of arginine de novo synthesis despite a significantly increased arginine turnover (flux), suggesting that this amino acid is a conditionally indispensable amino acid after major burns. This study used [15N2-guanidino-5,5-2H2]arginine and [5-13C]ornithine as tracers to assess the rate of arginine disposal via its conversion to and subsequent oxidation of ornithine; [5,5-2H2]proline and [5,5,5-2H3]leucine were also used to assess proline and protein kinetics. Nine severely burned patients were studied during a protein-free fast ("basal" or fast) and total parenteral nutrition (TPN) feedings. Compared with values from healthy volunteers, burn injury significantly increased 1) fluxes of arginine, ornithine, leucine, and proline; 2) arginine-to-ornithine conversion; 3) ornithine oxidation; and 4) arginine oxidation. TPN increased arginine-to-ornithine conversion and proportionally increased irreversible arginine oxidation. The elevated arginine oxidation, with limited net de novo synthesis from its immediate precursors, further implies that arginine is a conditionally indispensable amino acid in severely burned patients receiving TPN.     

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1‐naphthylalanine or 2‐naphthylalanine

We designed five ascidiacyclamide analogues [cyclo(‐Xxx¹‐oxazoline²‐d ‐Val³‐thiazole⁴‐l ‐Ile⁵‐oxazoline⁶‐d ‐Val⁷‐thiazole⁸‐)] incorporating l ‐1‐naphthylalanine (l ‐1Nal), l ‐2‐naphthylalanine (l ‐2Nal), d ‐phenylalanine (d ‐Phe), d ‐1‐naphthylalanine (d ‐1Nal) or d ‐2‐naphthylalanine (d ‐2Nal) into the Xxx¹ position of the peptide. The conformations of these analogues were then examined using ¹H NMR, CD spectroscopy, and X‐ray diffraction. These analyses suggested that d ‐enantiomer‐incorporated ASCs [(d ‐Phe), (d ‐1Nal), and (d ‐2Nal)ASC] transformed from the folded to the open structure in solution more easily than l ‐enantiomer‐incorporated ASCs [(l ‐Phe), (l ‐1Nal), and (l ‐2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1‐naphthyl isomer induced a more stable open structure than the 2‐naphthyl isomer. In particular, [d ‐1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL‐60 cells. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of arginine transport in Helicobacter pylori

Background. The amino acid L‐arginine is an essential requirement for growth of Helicobacter pylori. Several physiological roles of this amino acid have been identified in the bacterium, but very little is known about the transport of L‐arginine and of other amino acids into H. pylori. Methods. Radioactive tracer techniques using L‐(U‐¹⁴C) arginine and the centrifugation through oil method were employed to measure the kinetic parameters, temperature dependence, substrate specificity, and effects of analogues and inhibitors on L‐arginine transport. Results. The transport of arginine at millimolar concentrations was saturable with a K_m of 2.4 ± 0.3 mM and V_max of 1.3 ± 0.2 pmole min^?1 (µl cell water)^?1 or 31 ± 3 nmole per minute (mg protein)^?1 at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors. These characteristics suggested the presence of one or more arginine carriers. The substrate specificity of the transport system was studied by measuring the effects of L‐arginine analogues and amino acids on the rates of transport of L‐arginine. The absence of inhibition in competition experiments with L‐lysine and L‐ornithine indicated that the transport system was not of the Lysine‐Arginine‐Ornithine or Arginine‐Ornithine types. The presence of different monovalent cations did not affect the transport rates. Several properties of L‐arginine transport were elucidated by investigating the effects of potential inhibitors. Conclusions. The results provided evidence that the transport of L‐arginine into H. pylori cells was carrier‐mediated transport with the driving force supplied by the chemical gradient of the amino acid.     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.