Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.     

Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-(14)C]- or [2-(14)C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of (14)C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C(3) unit derived directly from lactate with a C(1) unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA-->crotonyl-CoA-->glutaconate-->glutamate.     

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.     

A New Pathway to Aspartic Acid from Urea and Maleic Acid Affected by Ultraviolet Light

The photochemistry of a mixture of ureaand maleic acid, which are thought to have been widelypresent on the primitive Earth, was studied in order toexamine a possibility of the formation of amino acids. When an aqueous solution of urea and maleic acid wasirradiated with an ultraviolet light of wavelength 172 nm,urea was revealed to be rather resistant to photochemicaldecomposition. In contrast, maleic acid was completelydecomposed within 4 h, reflecting the reactivity of a C-Cdouble bond in the molecule. In the reaction mixture, 2-isoureidosuccinic acid was detected. The acid wasconsidered to be formed by addition of an isoureido radicalwhich had been produced from urea by the action of ahydroxyl radical, to a C-C double bond of maleic acid. Theisoureido group of the product was revealed to undergothermal rearrangement to afford 2-ureidosuccinic acid (N-carbamoylaspartic acid). The result suggested a novelpathway leading to the formation of aspartic acid from non-amino acid precursors, possibly effected by UV-light on theprimitive Earth. The formation of ureidocarboxylic acidsis of another significance, since they are capable ofundergoing thermal polymerization, resulting in formationof polyamino acids.     

Thermal proteinoids as excitability-inducing materials

The deposition of thermal copolyamino acids on planar lipid membranes causes oscillations and action potentials upon electrical stimulation. Results are reported for compositionally simple thermal copoly(asp,glu) and for a more heterotonic polyamino acid. The data conform to the interference that electrical activity of cellular membranes is due to the polypeptide components, not to the lipid components. Because of the ease and controllability of producing polypeptides by thermal copolymerization of amino acids, new possibilities in investigation of structure-excitability relationships are provided.     

Poly(Aspartic Acid) Degradation by a Sphingomonas sp. Isolated from Freshwater

A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer.     

Some analogies between prebiotic thermal conversions of glycine and metabolic pathways

The reaction schemes suggested earlier for thermal transformation of glycine into amino acids and carboxylic acids are considered in detail. Close analogy with some wide-spread biochemical reactions of amino acids is observed. The pathway suggested has some common stages with the tricarboxylic acid cycle and other metabolic processes. The possible role of alpha-imino or alpha-keto acids as prebiological analogs of pyridoxal-phosphate-containing enzymes is discussed. The thermal transformations of glycine under primitive Earth conditions could be considered as evolutionary precursors of some present-day metabolic pathways.     

Activation of carboxyl group with cyanate: peptide bond formation from dicarboxylic acids

The reaction of cyanate with C-terminal carboxyl groups of peptides in aqueous solution was considered as a potential pathway for the abiotic formation of peptide bonds under the condition of the primitive Earth. The catalytic effect of dicarboxylic acids on cyanate hydrolysis was definitely attributed to intramolecular nucleophilic catalysis by the observation of the ¹H-NMR signal of succinic anhydride when reacting succinic acid with KOCN in aqueous solution (pH 2.2–5.5). The formation of amide bonds was noticed when adding amino acids or amino acid derivatives into the solution. The reaction of N-acyl aspartic acid derivatives was observed to proceed similarly and the scope of the cyanate-promoted reaction was analyzed from the standpoint of prebiotic peptide formation. The role of cyanate in activating peptide C-terminus constitutes a proof of principle that intramolecular reactions of adducts of peptides C-terminal carboxyl groups with activating agents represent a pathway for peptide activation in aqueous solution, the relevance of which is discussed in connexion with the issue of the emergence of homochirality.     

Polycondensation of thermal precursors of amino acids and characterization of constituent amino acids.

As a model reaction of polyamino acid formation from non-amino acid precursors, diammonium citraconate (I), ammonium citraconamate(II) and ammonium itaconamate(III) were converted to polyimide type polymers by thermal polycondensation by heating at 130–210°C. The imide type polymer was partially hydrolyzed to the corresponding peptide type polyamino acid. The polymer was composed of α-methylaspartic acid (IV), threo- and erythro-ß-methylaspartic acid (V) and α-(aminomethyl) succinic acid (VI). On the other hand, IV was thermally polycondensed to the corresponding polymer. It was found that the amino acid composition of the polymer was similar to that of the polymer prepared from I, II and III. The formation and isomerization of amino acids during the thermal polycondensation are described.     

The formation of chromophores through amino acid thermolysis and their possible role as prebiotic photoreceptors

The thermal polymerization of amino-acid mixtures was studied at various temperatures and reaction times with specific emphasis on the formation of fluorescent chromophores. The reaction conditions appeared to have a pronounced effect on the ratio of synthesized chromophores and biuret-positive material. During thermolysis of equimolar mixtures of lysine, alanine and glycine or lysine, aspartic acid and glycine small amounts of pteridines and flavines are formed, which are often covalently linked to the thermal oligomer. These heterocyclic compounds are likely formed by condensation reactions of the amino acid break-down and conversion products. Reaction schemes that describe the processes are proposed. The significance of these chromoproteinoids is discussed in respect to prebiotic redox reactions and photoinduced processes.     

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.     

A study of the interaction of glycine and its oligohomopeptides with formaldehyde and acetaldehyde under possible primitive earth conditions

It is established that glycine and glycine oligohomopeptides interact with formaldehyde and acetaldehyde in a homogeneous weak acid medium (pH 3.3–3.7) at mild temperatures (60–80°C) in the absence of inorganic solid substances. Together with the expected serine and threonine, the formation of alanine, glutamic and aspartic acid, norvaline and isoleucine, as well as four non-protein amino acids is also established. It is suggested that the non-protein amino acids are hydroxymethylserine, hydroxymethylthreonine, hydroxymethylaspartic acid and γ-amino-δ-hydroxyvaleric acid. The modes of formation of all protein and non-protein amino acids are discussed. These results strengthen the probability that similar processes may have been one of the pathways for the prebiotic synthesis of amino acids on primitive Earth.     

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.     

The biosynthesis of tabtoxinine-beta-lactam. Use of specifically 13C-labeled glucose and 13C NMR spectroscopy to identify its biosynthetic precursors

Tabtoxinine-beta-lactam, an irreversible inhibitor of glutamine synthetase is produced by several pathovars of Pseudomonas syringae. We have examined tabtoxinine-beta-lactam biosynthesis, an important and poorly characterized step in pathogenesis caused by this organism. We have identified the biosynthetic precursors of tabtoxinine-beta-lactam by incorporating 13C from specifically 13C-labeled D-glucose precursors and determining the labeling pattern using 13C NMR spectroscopy. Tabtoxinine-beta-lactam is generated by combining a 4-carbon fragment, a 2-carbon fragment, and a single carbon. The 4-carbon fragment arises from aspartic acid, and the 2-carbon unit is donated from carbons 2 and 3 of pyruvate. The 6-carbon backbone of tabtoxinine-beta-lactam arises from the condensation of fragments from aspartate and pyruvate, probably using reactions analogous to the initial steps in the pathway of lysine biosynthesis.     

Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic acids: cross-coupling and dimerization reactions

An anionic potato peroxidase (EC 1.11.1.7, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively.     

Production of Hexanal and Ethane by Phaeodactylum triconutum and Its Correlation to Fatty Acid Oxidation and Bleaching of Photosynthetic Pigments

In a light-dependent reaction (3.5 kilolux) at pH 5, the evolution of hexanal, ethane, and ethylene has been established with cell suspensions of the diatom, Phaeodactylum tricornutum. During this process, chlorophyll and carotenoids are partially bleached. Addition of 25 millimolar α-linolenic acid or 12 millimolar docosahexaenoic acid yield total pigment destruction and enhancement of ethylene and ethane formation (by about 150 and 7,600%, respectively), whereas hexanal production decreases by 70%. Eicosapentaenoic acid, the major polyunsaturated fatty acid in diatoms, stimulates both ethane and hexanal formation (by about 1,400 and 130%, respectively), but reduces ethylene production (by about 60%). This competition suggests that the production of the volatile compounds is closely connected, although hexanal and ethylene obviously possess different unsaturated fatty acids as precursors. Both the kind of the fatty acids and their relative amounts seem to determine the pattern of the evolved hydrocarbons. The presence of 10 millimolar propylgallate inhibits the evolution of the volatile compounds by about 80%, indicating that radical formation might play a key role in this light-dependent cascade of reactions.     

The Nicotinamide Biosynthetic Pathway Is a By-Product of the RNA World

Many of the biosynthetic pathways, especially those leading to the coenzymes, must have originated very early, perhaps before enzymes were available to catalyze their synthesis. While a number of enzymatic reactions in metabolism are known to proceed nonenzymatically, there are no examples of entire metabolic sequences that can be achieved in this manner. The most primitive pathway for nicotinic acid biosynthesis is the reaction of aspartic acid with dihydroxyacetone phosphate. We report here that nicotinic acid (NAc) and its metabolic precursor, quinolinic acid (QA), are produced in yields as high as 7% in a six-step nonenzymatic sequence from aspartic acid and dihydroxyacetone phosphate (DHAP). The biosynthesis of ribose phosphate could have produced DHAP and other three carbon compounds. Aspartic acid could have been available from prebiotic synthesis or from the ribozyme synthesis of pyrimidines. These results suggest that NAD could have originated in the RNA world and that the nonenzymatic biosynthesis of the cofactor nicotinamide could have been an inevitable consequence of life based on carbohydrates and amino acids. The enzymes of the modern pathway were later added in any order. Received: 22 May 2000 / Accepted: 7 August 2000     

Cucujolide biosynthesis in the merchant and rusty grain beetles

Most known aggregation pheromones of cucujid grain beetles are macrolides called “cucujolides”. It has recently been shown by us that cucujolide I[4(E),8(E)-4,8-dimethyldecadien-10-olide] is of terpenoid origin, and that cucujolide II[3(Z)-dodecen-11-olide] is of fatty acid. The objectives of this study were to determine if farnesol could serve as a precursor of cucujolide I in vivo; to study the conversion of fatty acids to cucujolide II; and to study the stereochemistry of the lactonization reactions leading to cucujolides I and II. Experiments were performed through application of stable isotope-labeling techniques, using the merchant grain beetle, Oryzaephilus mercator (Fauvel), and/or the rusty grain beetle, Cryptolestes ferrugineus (Stephens), as study insects. Exogenous (E,E)-farnesol was converted to cucujolide I. Dual-labeling studies with D and ¹⁸O indicate that this conversion proceeded with retention of the hydroxyl oxygen. Lauric and 11-hydroxydodecanoic acids were not effective precursors of cucujolide II, whereas 3(Z)-dodecenoic acid and 11-hydroxy-3(Z)-dodecenoic acid were effective precursors of cucujolide II. These data support the hypothesis that the biosynthetic route from fatty acids to cucujolide II involves chain shortening through β-oxidation to a 3(Z)-dodecenoic acid intermediate and oxidation at carbon-11 to form a 11-hydroxy-3(Z)-dodecenoic acid intermediate, followed by cylization. Dual-labeling studies with D and ¹⁸O indicate that this cyclization proceeded with retention of the C-11 hydroxyl of the 11-hydroxy-3(Z)-dodecenoic acid intermediate.     

Growth Retardation, Impaired Triacylglycerol Catabolism, Hepatic Steatosis, and Lethal Skin Barrier Defect in Mice Lacking Comparative Gene Identification-58 (CGI-58)

Comparative gene identification-58 (CGI-58), also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause “neutral lipid storage disease” characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58^−/−) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58^−/− mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.