Production of d-proline from l-arginine using Pseudomonas aeruginosa

Several microorganisms that can use (S)-5-[(amino-iminomethyl) amino]-2-chloropentanoic acid (l-Cl-arginine) as a nitrogen source have been isolated, the most interesting of which is a spontaneous mutant of Pseudomonas aeruginosa PAO1 (DSM 10581). In a fermenter, this unique biocatalyst hydrolysed l-Cl-arginine to (S)-5-amino-2-chloropentanoic acid (l-Cl-ornithine), which spontaneously converted to d-proline with inversion of configuration at an apparent average rate of 0.12 mmol ^−l h⁻¹ OD⁻¹. The enzyme, for which we suggest the name Cl-arginine amidinohydrolase, was best induced by using the substrate l-Cl-arginine as inducer and l-arginine as nitrogen source. The results presented here describe a new route for the production of d-proline from l-arginine, involving a chemical step and a biocatalytic step followed by a spontaneous chemical cyclisation.     

Improvement of the intracellular environment for enhancing l-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply

l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H₂O₂-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H₂O₂ synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3 g/L and 0.326 g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H₂O₂ synthesis and increasing the ATP supply.     

Isolation and characterization of a stable l-arginine producer from continuous culture broth of Corynebacterium acetoacidophilum

In order to produce l-arginine efficiently, continuous culture was attempted using an l-arginine producing strain of Corynebacterium acetoacidophilum, MC-13. l-Arginine production by strain MC-13 decreased after shifting from fed-batch culture to continuous culture because various strains with reduced or no l-arginine productivity, including l-arginine auxotrophs, appeared in addition to the original l-arginine producer. Strain SC-190, isolated from the continuous culture broth of strain MC-13, produced l-arginine on cultivation in a stable fashion for more than 250 h. It is suggested that strain SC-190 acquired a lower susceptibility to the inhibition of growth and l-arginine production by l-arginine than the parental strain MC-13 due to this stabilization.     

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Structural and mechanistic studies on N-(2-carboxyethyl)arginine synthase

N²-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the β-lactamase inhibitor clavulanic acid in Streptomyces clavuligerus. Crystal structures of tetrameric CEAS-ThDP in complex with the substrate analogues 5-guanidinovaleric acid (GVA) and tartrate, and a structure reflecting a possible enol(ate)-ThDP reaction intermediate are described. The structures suggest overlapping binding sites for the substrates d-glyceraldehyde-3-phosphate (d-G3P) and l-arginine, and are consistent with the proposed CEAS mechanism in which d-G3P binds at the active site and reacts to form an α,β-unsaturated intermediate, which subsequently undergoes (1,4)-Michael addition with the α-amino group of l-arginine. Additional solution studies are presented which probe the amino acid substrate tolerance of CEAS, providing further insight into the l-arginine binding site. These findings may facilitate the engineering of CEAS towards the synthesis of alternative β-amino acid products.     

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from ¹⁴C-mevalonic acid or ¹⁴C-acetate, permitting preparation of ¹⁴C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.     

Survey of <Emphasis Type="Italic">Alternaria</Emphasis> toxin contamination in food from the German market,using a rapid HPLC-MS/MS approach

A HPLC-MS/MS-based method for the quantification of nine mycotoxins produced by fungi of the genus Alternaria in various food matrices was developed. The method relies on a single-step extraction, followed by dilution of the raw extract and direct analysis. In combination with an analysis time per sample of 12 min, the sample preparation is cost-effective and easy to handle. The method covers alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), iso-altenuene (isoALT), tentoxin (TEN), altertoxin-I (ATX-I), and the AAL toxins TA₁ and TA₂. Some Alternaria toxins which are either not commercially available or very expensive, namely AOH, AME, ALT, isoALT, and ATX-I, were isolated as reference compounds from fungal cultures. The method was extensively validated for tomato products, bakery products, sunflower seeds, fruit juices, and vegetable oils. AOH, AME, TeA, and TEN were found in quantifiable amounts and 92.1 % of all analyzed samples (n?=?96) showed low level contamination with one or more Alternaria toxins. Based on the obtained results, the average daily exposure to Alternaria toxins in Germany was calculated.     

Transport of l-arginine in brush border vesicles derived from rabbit kidney cortex

The uptake of l-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37 °C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K_m values of 0.07 and 3.5 mm and V_max values of 1.5 and 8 nmol/mg protein × 15 s, respectively. For the inhibitors of l-[¹⁴C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mm in a mannitol medium) the following sequence of inhibitory strength was established: l-arginine, l-ornithine, l-cystine, l-lysine, d-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K⁺ in the presence of gramicidin D, an overshoot of l-arginine uptake was observed. Initial uptake of l-arginine was slightly faster in the presence of a Na⁺ gradient (outside to inside) than under a K⁺ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, l-arginine uptake in the first 30 s was faster in the NaCl than in the KCl medium. These experiments indicate, beside a major ion-independent l-arginine transport, the presence of a transport stimulated by Na⁺ in isolated brush border vesicles.     

New weak toxins from the cobra venom

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin, and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by α7 and muscle-type nicotinic acetylcholine receptors.     

Heat measurements applied to biochemical analysis: glucose in human serum.

d-(?)-Arginine was isolated by fractionally recrystallizing dl-arginine-l_s-(?)-malate to obtain the less soluble d-(?)-arginine-l_s-(?)-malate, then passing aqueous solutions of the latter through ionic resin columns to separate the d-arginine from the malic acid. The d-arginine was converted to the monohydrochloride. The fractional crystallization readily yielded 51–73% of the d-arginine-l-malate. d-Arginine monohydrochloride was obtained from the latter in yields of 74–83% of the theoretical.Impure l-arginine was separated from the more soluble arginine malate residues. Two methods of racemizing it for recycling were tested. The separated malic acid was evaporated to dryness in vacuo for reuse, or precipitated as the calcium salt.     

Dietary l-arginine supplementation improves semen quality and libido of boars under high ambient temperature

l-Arginine is a nutritionally essential amino acid for spermatogenesis and plays versatile roles in animal health and can be utilized as a potential agent to improve reproductive performance of boars under high ambient temperature. The present study aimed to determine whether dietary l-arginine could alleviate heat stress-induced infertility in boars. In all, 20 boars (PIC 1040; 248.59±3.84 kg BW and 407.65±6.40 days of age) were selected and randomly assigned to four groups (group 0.0%, basal diet; group 0.6%, 0.8% or 1.0%, basal diet added with 0.6%, 0.8% or 1.0% l-arginine (wt:wt), respectively.) The four diets were made isonitrogenous by addition of appropriate amounts of l-alanine. Boars were pre-fed the corresponding experimental diet for 42 days. Then, the semen characteristics and libido were accessed for 6 weeks during the hot summer period (25.5° to 33.0°C). Results show that dietary l-arginine remarkably improved sperm motility, normality, total sperm number and effective total sperm number. Also, dietary l-arginine improved semen antioxidant capacity, such as decrease of malondialdehyde and 8-Hydroxy-2'-deoxyguanosine content in sperm (P<0.05), increase of the ratio of glutathione and oxidized glutathione, total antioxidant capacity, glutathione peroxidase and catalase activities in seminal plasma (P<0.05). Most of mitochondria contained intact ultrastructure in l-arginine-supplemented group which also accompany with higher ATP content than the 0.0% group. The boars fed 0.8% l-arginine show increased levels of estradiol-17β and testosterone and exhibit improved libido performance than boars in the 0.0% group. Adding dietary l-arginine linearly increased (P=0.002) nitric oxide content (as l-arginine increased). The scrotal surface temperature in the 0.6%, 0.8% and 1.0% group were decreased by 0.9°C, 0.9°C and 0.4°C, respectively, compared with the 0.0% group. l-Arginine levels caused linear effect on semen quality and antioxidant capacity, also caused quadratic effect on libido performance. During the hot summer months, the predicted optimal l-arginine levels for best semen quality and antioxidant capacity was 0.8% to 1.0% and for best libido performance was 0.8%. It can be concluded that l-arginine can be used as an effective agent to alleviate heat stress-induced infertility of boar, and that 0.8% to 1.0% can be considered as the optimum dosage.     

Effects of dietary l-arginine supplementation to gilts during early gestation on foetal survival, growth and myofiber formation

The effects of l-arginine on porcine foetal development and myogenesis were determined. Twenty Swiss Large White gilts were randomly allocated to either the control (C) or l-arginine treatment (A). In addition to the standard gestation diet, A-sows received 26 g l-arginine daily from days 14 to 28 of gestation. At day 75 of pregnancy, sows were sacrificed and the number and weight of foetuses were recorded. From each litter, the lightest, heaviest and the ones with an average foetal weight (FtW) were selected. Primary (P), secondary (S) and total myofiber number as well as S/P ratio were determined in the semitendinosus (ST) and rhomboideus (RH) muscles. In A-sows, the number of viable foetuses (13.0 v. 9.3) and total FtW (4925 v. 3729 g) was greater (P ⩽ 0.04) than in C-sows. Compared to C-sow foetuses, the ST of A-sow foetuses had 7% more (17 699 v. 16 477; P = 0.04) P myofibers and the S/P ratio in both muscles was lower (ST = 20.3 v. 21.5; RH = 24.1 v. 27.1; P ⩽ 0.07). Regardless of the maternal diet, the S myofiber number and the S/P ratio in both muscles were greater (P ⩽ 0.01) in foetuses with a high FtW compared to low FtW. These data suggest that l-arginine supplemented to gilts during early gestation enhanced foetal survival and in the ST positively affected the primary phase of myofiber formation.     

Contamination of Bananas with Beauvericin and Fusaric Acid Produced by Fusarium oxysporum f. sp. cubense

Background

Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis.

Methodology/Principal Findings

Twenty isolates of Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak ‘Guangfen #1’ and 10 Cavendish ‘Brazilian’ plants. Fusaric acid and BEA were detected in all the tissues, including the fruits.

Conclusions/Signficance

The current study provides the first investigation of toxins produced by Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.     

Plasma l-arginine is markedly reduced in pregnant women affected by preeclampsia

The objective of this study was to determine the concentration of free l-amino acids and in particular of l-arginine in the plasma of pregnant women affected by preeclampsia compared to healthy pregnant women in order to know if an alteration in the concentrations of these amino acids occurs in preeclamspia. Twelve pregnant women affected by preeclampsia and twelve pregnant control women, ages 28–35 years old and at the 35–36 weeks of pregnancy were studied. The blood analysis of free amino acids was carried out by using a high performance liquid chromatographic (HPLC) fluorometric method and OPA-NAC as derivatizing agent for the amino acid determination. In the blood of women affected by preeclampsia l-arginine is markedly reduced compared to controls (about five-fold lower, P<0.01). The other amino acids also are significantly reduced, but to lesser extents (about 1.5 times lower, P<0.05). Thus, the determination of l-arginine in the blood of pregnant women could potentially constitute an additional marker for the early diagnosis of preeclampsia.     

l-Arginine uptake into renal brush border membrane vesicles

The uphill uptake of l-arginine by renal brush border membrane vesicles was found to be energized by a Na⁺ gradient (extravesicular > intravesicular) in the presence of a membrane potential (inside negative). The uptake was specific for Na⁺. Either a K⁺-diffusion potential, generated by valinomycin, or a H⁺-diffusion potential, generated by the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, provided the electrical driving force. The Na⁺ gradient-dependent l-arginine transport system was shared by specific basic amino acids and l-cystine, but not by d-arginine nor other classes of amino acids. The molecular structure of the basic amino acid recognized by the carrier was postulated.     

Factors affecting l-arginine production in the continuous culture of an l-arginine producer of Corynebacterium acetoacidophilum

The effects of culture conditions on l-arginine production by continuous culture were studied using a stable l-arginine hyperproducing strain of Corynebacterium aceto-acidophilum, SC-190. Strain SC-190 demonstrated a volumetric productivity of 35 g l⁻¹·h⁻¹ at a dilution rate of 0.083h⁻¹ and feeding sugar concentration of 8%, and a product yield of 29.2% at a dilution rate 0.021h⁻¹ and feeding sugar concentration of 15%. The corresponding values for fed-batch culture are 0.85 g·l⁻¹·h⁻¹ and 26%. However, the product yield decreased with an increase in the volumetric productivity. To achieve stable l-arginine production, aeration and agitation conditions sufficient to maintain an optimal level of redox potential (>−100 mV) were necessary. The addition of phosphate to the feeding medium led to a decrease in l-arginine production. It was confirmed in the steady state that growth and l-arginine formation were inhibited by a high concentration of l-arginine.     

A structural basis for substrate selectivity and stereoselectivity in octopine dehydrogenase from Pecten maximus

Octopine dehydrogenase [N²-(d-1-carboxyethyl)-l-arginine:NAD⁺ oxidoreductase] (OcDH) from the adductor muscle of the great scallop Pecten maximus catalyzes the reductive condensation of l-arginine and pyruvate to octopine during escape swimming. This enzyme, which is a prototype of opine dehydrogenases (OpDHs), oxidizes glycolytically born NADH to NAD⁺, thus sustaining anaerobic ATP provision during short periods of strenuous muscular activity. In contrast to some other OpDHs, OcDH uses only l-arginine as the amino acid substrate. Here, we report the crystal structures of OcDH in complex with NADH and the binary complexes NADH/l-arginine and NADH/pyruvate, providing detailed information about the principles of substrate recognition, ligand binding and the reaction mechanism. OcDH binds its substrates through a combination of electrostatic forces and size selection, which guarantees that OcDH catalysis proceeds with substrate selectivity and stereoselectivity, giving rise to a second chiral center and exploiting a “molecular ruler” mechanism.     

Cysteine-rich toxins from Lachesana tarabaevi spider venom with amphiphilic C-terminal segments

Venom of Lachesana tarabaevi (Zodariidae, “ant spiders”) exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices.     

A procedure for the enrichment and isolation of Halobacterium

A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of l-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community.