Enzyme organization in the proline biosynthetic pathway of Escherichia coli

The conversion of glutamic acid to proline by an Escherichia coli extract was studied. The activity was dependent upon the presence of ATP and NADPH and was largely unaffected by the presence of NH₃ or imidazole. The first two pathway enzymes appear to exist as a complex which stabilizes a labile intermediate postulated as γ-glutamyl phosphate. Attempted synthesis of this compound was unsuccessful due to its spontaneous cyclization to 2-pyrrolidone 5-carboxylate. Dissociation of the enzyme complex upon dilution of the extract is presumed responsible for an experimentally observed “dilution effect”. E. coli pro_A^? and pro_B^? auxotroph extracts failed to complement one another in the biosynthesis of proline. This is attributed to the lack of a dynamic equilibrium between the complex and its constituent enzymes.In vivo studies with E. coli showed no evidence for metabolic channeling in the final reaction of proline synthesis, the reduction of Δ¹-pyrroline 5-carboxylate.     

Pyrroline-5-Carboxylate Reductase in Soybean Nodules : Comparison of the Enzymes in Host Cytosol, Bradyrhizobium japonicum Bacteroids, and Cultures

Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD⁺-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP⁺ and proline. The K₁ for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable.     

Δ1-Pyrroline-5-carboxylate: The product of proline dehydrogenase from Cucurbita moschata cotyledons

The product of oxidation of proline by pumpkin proline dehydrogenase reacted with o-aminobenzaldehyde to give a yellow compound that had an absorption spectrum similar to that obtained from chemically synthesized Δ¹-pyrroline-5-carboxylate. The product of the proline dehydrogenase reaction and synthetic Δ¹-pyrroline-5-carboxylate had identical R_f values. Both authentic Δ¹-pyrroline-5-carboxylate and the product of the enzyme gave a pink colour with acid ninhydrin on paper chromatograms and both had identical elution patterns on Dowex 50(H⁺) columns. Neither synthetic Δ¹-pyrroline-5-carboxylate nor the product of proline-dehydrogenase produced γ-amino butyrate with hydrogen peroxide.     

Modulation of 2-Acetyl-1-pyrroline (2-AP) Accumulation,Yield Formation and Antioxidant Attributes in Fragrant Rice by Exogenous Methylglyoxal (MG) Application

Pot experiments were conducted to investigate the physiological and biochemical effect of exogenous MG application on two fragrant rice cultivars, Meixiangzhan and Yuxiangyouzhan. The MG application included four treatments: 1 mmol·L^–1 (MG1), 5 mmol·L^–1 (MG5), and 10 mmol·L^–1 (MG10), and deionized water as control (MG0). Results indicated that MG1 significantly increased the yield by 11.54–14.06% through promoting the seed-setting rate. Increases on grain 2-acetyl-1-pyrroline (2-AP) content at maturity were found in MG1. The contents of proline, Δ1-pyrrolin-5-carboxylic acid (P5C), pyrroline, and MG, and the activities of proline dehydrogenase (PDH), ornithine aminotransferase (OAT), and Δ1-pyrrolin-5-carboxylate synthase (P5CS) in plant tissues at all sampling stage were also increased in MG1 than MG0. In addition, although the malondialdehyde (MDA) content at all sampling stage was increased by MG application, MG1 increased the antioxidant enzyme activities and soluble protein content in all plant tissues and the pigment content in leaves. However, high-level MG (10 mmol·L^–1) application significantly decreased the yield and grain 2-AP content. Overall, this study suggested that exogenous MG application regulated the 2-AP accumulation, yield formation, and antioxidant attributes in fragrant rice, and that MG application at the concentration of 1 mmol·L^–1 increased the 2-AP content.

     

The importance of Δ1-pyrroline in the aetiology of cerebrocortical necrosis

Bacterial thiaminase I associated with cerebrocortical necrosis of cattle and sheep is shown to utilise Δ¹-pyrroline and related compounds as cosubstrates. The product resulting from the reaction of thiamine and Δ¹-pyrroline is 1-(4-amino-2-methylpyrimidin-5-ylmethyl)-1-pyrrolinium chloride. This compound has been identified in the brains of calves suffering from cerebrocortical necrosis. The implications of these findings in the aetiology of other thiamine-responsive diseases of the central nervous system are briefly discussed.     

Periodate oxidation products of hydroxylysine in the synthesis of 5-substituted prolines

The amino acid hydroxylysine was subjected to oxidation by sodium metaperiodate under various conditions. It was found that in acid and high temperature, the initial oxidation product alpha-aminoglutaric gamma-semialdehyde was converted to glutamic acid with a yield of 60%. The use of alkaline conditions of oxidation favored the cyclization of alpha-aminoglutaric gamma-semialdehyde to form delta 1-pyrroline 5-carboxylic acid. Addition of NaCN to this intermediate generated new proline analogs, likely a mixture of cis- and trans-5-cyanoprolines, with a yield of 30%. Upon hydrolysis, the 5-cyanoprolines were converted to a probable mixture of cis- and trans-5-carboxyprolines. Infrared and high-resolution mass spectral data of the analogs and visual absorption spectra of the ninhydrin products were obtained to confirm the structures.     

Pyrroline-5-carboxylic acid reductase from soybean leaves

Pyrroline-5-carboxylic acid reductase was purified 40-fold from soybean leaves (Glycine max L. var Corsoy). The enzyme was fairly unstable, had a broad pH optimum, and was inactivated by heat and acid; NADH and NADPH both served as cofactors. It had a higher activity with NADH (about 4 ×) compared to NADPH, but a lower K_m for NADPH. NADP⁺ inhibited both the NADH- and NADPH-dependent activity. Sulfhydryl group blocking agents reduced the activity as did the carbonyl blocking agent, NH₂OH. Thiazolidine-4-carboxylic acid and phosphate inhibited the enzyme and proline inhibited only at high concentrations. ATP, GTP, and CTP were all effective inhibitors of both the NADH- and NADPH-dependent activity. Phosphorylated nucleotide inhibition was reversed by Mg²⁺ ions.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Oxidation of proline by plant mitochondria

Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O₂ uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O₂, but not NAD. The major ninhydrin-positive product formed via Δ¹-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO₂, organic acids, and aspartate were recovered after [¹⁴C]proline feeding experiments. These results demonstrate the mitochondrial association and O₂ dependence of plant proline metabolism.     

Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria

L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780-784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K_m for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD⁺-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments.     

Isolation and characterization of a novel biosurfactant produced by hydrocarbon‐degrading bacterium Alcanivorax dieselolei B‐5

Aims: Our goal was to identify a novel biosurfactant produced by a marine oil‐degrading bacterium. Methods and Results: Biosurfactants were produced by Alcanivorax dieselolei strain B‐5^T growing with diesel oil as the sole carbon and energy source. Culture supernatant was first extracted with chloroform/methanol (1 : 1, v/v), then further purified step by step with a normal phase silica gel column, a Sephadex LH20 gel column and a preparative thin layer plate. The main component was determined to be a lipopeptide; it was chemically characterized with nuclear magnetic resonance, liquid chromatography‐quadrupole ion‐trap mass spectrometry, amino acid analysis and GC–MS and was found to be a mixture of proline lipids. The monomers of the proline lipids were composed of a proline residue and a fatty acid (C_14:0, C_16:0 or C_18:0). The critical micelle concentration of the mixed proline lipids was determined to be 40 mg l^?1. Moreover, activity variations in ranges of pH, temperature and salinity were also detected and showed reasonable stability. Conclusions: Alcanivorax dieselolei B‐5 produced a novel linear lipoamino biosurfactant, characterized as a proline lipid. Significance and Impact of the Study: A proline lipid was characterized for the first time as a bacterial biosurfactant. This product has potential in both environmental and industrial applications.     

Grain Yield,Quality and 2-Acetyl-1-Pyrroline of Fragrant Rice in Response to Different Planting Seasons in South China

Climate conditions is an important factor affected the fragrant rice growth and development. In order to study the effects of different planting seasons on fragrant rice performance in South China, present study was conducted with three planting seasons (early season (April to July), middle season (June to September) and late season (August to November)) and three fragrant rice cultivars, ‘Basmati-385’, ‘Meixiangzhan-2’ and ‘Xiangyaxiangzhan’. The results showed that the highest grain yield and grain 2-acetyl-1-pyrroline (2-AP, key component of fragrant rice aroma) content were both recorded in late season treatment while the fragrant rice in middle season treatment produced the lowest grain yield, grain filling percentage, 1000-grain weight and gain 2-AP content. The highest contents of precursors (proline, pyrroline-5-carboxylic acid and 1-pyrroline) which related to 2-AP biosynthesis were recorded in late season treatment compared with early season treatment and middle season treatment. The highest activities of enzymes (proline dehydrogenase, pyrroline-5-carboxylic acid synthetase and ornithine transaminase) which involved in 2-AP biosynthesis were also observed in late season treatment. Moreover, the fragrant rice cultivars in late season possessed the lowest chalk rice rate, chalkiness as well as the highest brown rice rate, head rice and protein content. Thus, the optimal season for fragrant rice production in South China is the late season.     

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl-3,4-Dehydroproline, l-α-azetidine-2-carboxylic acid and l-pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l-thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d- and l-isomers of 3,4-dehydroproline was compared with the racemic mixture; the l-isomer was twice as active as the latter, while the d-isomer was only half as active. l-3,4-Dehydroproline was approximately four times as potent as l-α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl-3,4-Dehydroproline inhibited the incorporation of l-[¹⁴C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [¹⁴C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl-3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo.These observations indicate that dl-3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl-3,4-dehydroproline (200 mg/kg).     

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0₂ uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited ¹-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.     

Proline as an intermediate in the reductive deamination of ornithine to delta-aminovaleric acid

Fresh extracts of cells of Clostridium botulinum reduced a limited amount of ornithine to delta-aminovaleric acid, but at high substrate concentrations a considerable amount of an amino compound accumulated which was neutral at pH 4.2. Aging of the extracts at -10 C or freezing and thawing resulted in the loss of the ability to produce delta-aminovaleric acid, but the ability to produce the neutral compound was retained. This compound was separated by column chromatography, and was found to be identical to dl-proline with respect to (i) R(F) upon paper chromatography, (ii) migration rates upon paper ionophoresis, (iii) spectrum of the product of the ninhydrin reaction, (iv) oxidation with d-amino acid oxidase, and (v) rate of reduction to delta-aminovaleric acid by cell extracts. The intermediate role of proline in the reduction of ornithine to delta-aminovaleric acid was indicated by (i) rate studies with and without an added electron donor and with and without inhibitors of proline reductase, (ii) the initial accumulation of radioactive proline to the exclusion of radioactive delta-aminovaleric acid from (14)C-l-ornithine in the presence of low levels of carrier proline, and (iii) the initial accumulation of proline at low levels prior to a significant accumulation of delta-aminovaleric acid in reaction mixtures in which the latter compound was the primary product after a longer incubation time. The conversion of ornithine to proline was the rate-limiting step in the presence of a good electron donor (alanine). The mechanism of the conversion of ornithine to proline has not been established. Preliminary data indicated that it may involve an oxidation to glutamic-gamma-semialdehyde and its equilibrium product, Delta(1)-pyrroline-5-carboxylic acid.     

Transfer of reducing equivalents into mitochondria by the interconversions of proline and Δ1-pyrroline-5-carboxylate

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers ³H from [1-³H]glucose to NADP⁺ to Δ¹-pyrroline-5-carboxylate and yields [³H]proline. The formation of [³H]proline depends on the presence of NADP, Δ¹-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ¹-pyrroline-5-carboxylate reductase. The production of [³H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ¹-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ¹-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ¹-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ¹-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.     

A simplified method for chromatography of Dnp-leucine in the determination of intracellular specific activity of (3H) leucine

We describe a radioisotopic assay for Δ¹-pyrroline-5-carboxylate reductase. In this assay we use Δ¹-pyrroline-5-carboxylate[U-¹⁴C] and isolate product l-[U-¹⁴C]proline by cation-exchange column chromatography.     

A rapid and sensitive colorimetric assay of amine oxidase

A sensitive colorimetric assay for amine oxidase with Δ¹-pyrroline as the product is described based on the formation of a red-colored complex with ninhydrin reagent in acidic medium.     

Effect of water stress on proline synthesis from radioactive precursors

Barley (Hordeum vulgare L. var. Prior) leaves converted more ¹⁴C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (Δ¹-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of ¹⁴C-glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility.     

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight