Nω-Hydroxyamino-α-amino acids as a new class of very strong inhibitors of arginases

 The effects of various compounds bearing an N-OH group such as N-hydroxy-guanidines, amidoximes, and hydroxylamines, on bovine and rat liver arginases was studied. Some of these compounds with an l-α-amino acid function at an appropriate distance from the N-OH group acted as strong competitive liver arginase inhibitors, displaying Ki values between 4 and 150 μM. Two compounds, N ^ε-hydroxy-l-lysine and N ^ω-hydroxy-d,l-indospicine, which exhibited Ki values of 4 and 20 μM (at pH 7.4), were the most potent inhibitors of arginase described to date. The distance between the α-amino acid and N-OH functions appeared to be crucial for potent inhibition of arginase, as N ^δ-hydroxy-l-ornithine, which has one -CH₂ group less than N ^ε-hydroxy-l-lysine, exhibited a 37-fold higher Ki value than N ^ε-hydroxy-l-lysine. Based on these results, a model for the interaction of N ^ω-hydroxyamino-l-α-amino acids with the arginase active site is proposed. This model involves the binding of the N-OH group of the inhibitors to the arginase Mn(II) center and suggests that N ^ε-hydroxy-l-lysine is a good transition state analog of arginase.     

D-amino acid N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free D-amino acids

d-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on d-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyltransferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of d-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except l-lysine and d-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of d-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

Eco-Efficiency Analysis of biotechnological processes

For almost 50 years now, biotechnological production processes have been used for industrial production of amino acids. Market development has been particularly dynamic for the flavor-enhancer glutamate and the animal feed amino acids l-lysine, l-threonine, and l-tryptophan, which are produced by fermentation processes using high-performance strains of Corynebacterium glutamicum and Escherichia coli from sugar sources such as molasses, sucrose, or glucose. But the market for amino acids in synthesis is also becoming increasingly important, with annual growth rates of 5–7%. The use of enzymes and whole cell biocatalysts has proven particularly valuable in production of both proteinogenic and nonproteinogenic l-amino acids, d-amino acids, and enantiomerically pure amino acid derivatives, which are of great interest as building blocks for active ingredients that are applied as pharmaceuticals, cosmetics, and agricultural products. Nutrition and health will continue to be the driving forces for exploiting the potential of microorganisms, and possibly also of suitable plants, to arrive at even more efficient processes for amino acid production.     

Nutritional and medicinal aspects of <Emphasis Type="SmallCaps">d</Emphasis>-amino acids

This paper reviews and interprets a method for determining the nutritional value of d-amino acids, d-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a nutritionally essential amino acid such as l-lysine (l-Lys), l-methionine (l-Met), l-phenylalanine (l-Phe), and l-tryptophan (l-Trp) as well as the semi-essential amino acids l-cysteine (l-Cys) and l-tyrosine (l-Tyr). The results show wide-ranging variations in the biological utilization of test substances. The method is generally applicable to the determination of the biological utilization and safety of any amino acid derivative as a potential nutritional source of the corresponding l-amino acid. Because the organism is forced to use the d-amino acid or amino acid derivative as the sole source of the essential or semi-essential amino acid being replaced, and because a free amino acid diet allows better control of composition, the use of all-amino-acid diets for such determinations may be preferable to protein-based diets. Also covered are brief summaries of the widely scattered literature on dietary and pharmacological aspects of 27 individual d-amino acids, d-peptides, and isomeric amino acid derivatives and suggested research needs in each of these areas. The described results provide a valuable record and resource for further progress on the multifaceted aspects of d-amino acids in food and biological samples.     

Effects of Glycine and d-Amino Acids on Growth of Various Microorganisms

Growth of various microorganisms in media containing high concentrations of glycine or d-amino acids was examined. Susceptibilities to glycine or d-amino acids differed among microorganisms, and the differences in susceptibility have no direct relation with Gram staining, morphological forms, and aerobic or anaerobic nature of the organisms. Certain glycine-resistant bacteria tested, which included Bacillus cereus, Staphylococcus aureus and Serratia marcescens, exhibited relatively high oxidative activities towards glycine. The inhibition of the growth of Escherichia coli by either glycine or d-amino acids, which included d-threonine, d-alanine and d-lysine, was reversed by l-alanine, partialy by l-serine, and not by l-lysine or l-threonine. These results suggest that the growth inhibition of microorganisms by d-amino acids was similar to that by glycine. The incorporation of l-alanine into E. coli cells which were preincubated with glycine was less than those of preincubated without glycine. Particularly, the incorporation into the cell wall fraction was most susceptible to glycine. An additive effect of penicillin and glycine was observed in the inhibition of cell wall biosynthesis as determined by the intracellular accumulation of N-acetylamino sugar compounds.     

d-Lysine production from l-lysine by successive chemical racemization and microbial asymmetric degradation

In order to develop a practical process for d-lysine production from l-lysine, successive chemical racemization and microbial asymmetric degradation were investigated. The racemization of l-lysine proceeded quantitatively at elevated temperatures. A sample␣of 1000 strains of bacteria, fungi, yeast and actinomyces were screened for the ability to degrade l-lysine asymmetrically. Microorganisms belonging to the Achromobacter, Agrobacterium, Candida, Comamonas, Flavobacterium, Proteus, Providencia, Pseudomonas and Yarrowia genera exhibited a high l-lysine-degrading activity. Comamonas testosteroni IAM 1048 was determined to be the best strain and used as a biocatalyst for eliminating the l isomer. The degradation rate of l-lysine with C. testosteroni IAM 1048 was influenced by pH, temperature and agitation speed. Under the optimal conditions, the l isomer in a 100-g/l mixture of racemic lysine was completely degraded within 72 h, with 47 g d-lysine/l left in the reaction mixture. Crystalline d-lysine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 38% from the reaction mixture by simple purification. An engineering analysis of l-lysine racemization and microbial degradation was carried out to establish the basis of process design for d-lysine production. Received: 24 September 1996 / Received last revision: 8 November 1996 / Accepted: 23 November 1996     

Chemotaxis and transport of amino acids in Allomyces arbuscula

Among a number of amino acids tested, l-lysine and l-arginine are the principal attractants in the chemotaxis of the zygotes of Allomyces arbuscula. The reaction can be stimulated to a greater or lesser extent by a number of compounds chemically related to l-leucine. No relationship between transport of attracting amino acids and their effect on chemotaxis has been found.     

Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids

Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.     

Carbamoylases: characteristics and applications in biotechnological processes

Enzymatic kinetic resolution is a widely used biotechnological tool for the production of enantiomerically pure/enriched compounds. This technique takes advantage of the enantioselectivity or enantiospecificity of an enzyme for one of the enantiomers of a racemic substrate to isolate the desired isomer. N-Carbamoyl-d- and l-amino acid amidohydrolases (d- and l-carbamoylases) are model enzymes for this procedure due to their strict enantiospecificity. Carbamoylase-based kinetic resolution of amino acids has been applied for the last three decades, allowing the production of optically pure d- or l-amino acids. Furthermore, this enzyme has become crucial in the industrially used multienzymatic system known as “Hydantoinase Process,” where the kinetic resolution produced by coupling an enantioselective hydantoinase and the enantiospecific carbamoylase is enhanced by the enzymatic/chemical dynamic kinetic resolution of the low-rate hydrolyzed substrate. This review outlines the properties of d- and l-carbamoylases, emphasizing their biochemical/structural characteristics and their biotechnological applications. It also pinpoints new applications for the exploitation of carbamoylases over the forthcoming years.     

Regulation of aspartokinase activity in non-fermentative,marine eubacteria

Summary Cell-free extracts of gram-negative, non-fermentative, marine eubacteria were assayed for aspartokinase activity. The organisms tested included polarly flagellated species and groups which had GC contents in their DNAs of 46 to 64 moles % (Alteromonas, Pseudomonas) as well as species which had peritrichous flagellation and moles % GC contents of 53 to 68 (Alcaligenes). The results of these studies suggested that in all the strains tested, aspartokinase activity was catalyzed by a single enzyme. On the basis of the effect ofl-threonine,l-lysine,l-methionine, andl-isoleucine on activity, five different types of aspartokinases (designated I through V) were delineated. In aspartokinase types I through IV,l-threonine andl-lysine inhibited activity by means of a concerted feedback inhibition; in type V, activity was inhibited byl-threonine but unaffected byl-lysine. In types I, III, and IV,l-threonine andl-lysine alone were inhibitory, while in type II these effectors had virtually no effect on activity when tested singly. Three distinct responses were observed in the presence of two other end products of the aspartate pathway,l-methionine andl-isoleucine. In types I and II, these two amino acids usually stimulated activity and overcame the inhibition byl-threonine andl-lysine; in types IV and V,l-methionine andl-isoleucine had no effect; and in type III these amino acids inhibited activity. The results of this study indicate that the aspartokinases of a number of species and groups of marine bacteria have similarities and differences which should be of use in making future taxonomic groupings.     

Uptake and conversion of <Emphasis Type="SmallCaps">d</Emphasis>-amino acids in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The d-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom, the metabolism and role of d-amino acids (d-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of d-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their d-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey’s reagent and separated by HPLC–MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied d-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of d-AAs. The addition of particular amino acids (d-Trp, d-Phe, d-Met and d-His) led to the accumulation of the corresponding l-amino acid. In almost all cases, the application of a d-AA resulted in the accumulation of d-Ala and d-Glu. The presented results indicate that soil borne d-AAs can actively be taken up and metabolized via central metabolic routes.     

The nitrogen requirements ofGluconobacter,Acetobacter andFrateuria

The nitrogen requirements of 96Gluconobacter, 55Acetobacter and 7Frateuria strains were examined. Only someFrateuria strains were able to grow on 0.5% yeast extract broth or 0.5% peptone broth. In the presence ofd-glucose ord-mannitol as a carbon source, ammonium was used as the sole source of nitrogen by all three genera. With ethanol, only a fewAcetobacter strains grew on ammonium as a sole nitrogen source. Singlel-amino acids cannot serve as a sole source of carbon and nitrogen for growth ofGluconobacter, Acetobacter orFrateuria. The singlel-amino acids which were used by most strains as a sole nitrogen source for growth are: asparagine, aspartic acid, glutamine, glutamic acid, proline and alanine. SomeAcetobacter andGluconobacter strains deaminated alanine, asparagine, glutamic acid, threonine, serine and proline. NoFrateuria strain was able to develop on cysteine, glycine, threonine or tryptophan as a sole source of nitrogen for growth. An inhibitory effect of valine may explain the absence of growth on this amino acid. No amino acid is “essential” forGluconobacter, Acetobacter orFrateuria.     

Light and dark metabolism ofl- andd-amino acids in 5 strains ofChlorella vulgaris and 4 strains of the genusAnkistrodesmus

In the light, 2 out of the 4 newly testedChlorella vulgaris strains were found to use about as many amino acids as a source of nitrogen as the previously investigated strain Delft; the other 2C. vulgaris strains and 3 of theAnkistrodesmus strains used only a few. The 4thAnkistrodesmus strain used none.On the average,l-amino acids supported better growth thand-amino acids, butd-serine was preferred tol-serine by 3Ankistrodesmus strains.In the dark, growth was only obtained withC. vulgaris strain Delft, and only on a few of thel-amino acids,l-leucine in particular.The author is indebted to the Direction of the Academic Hospital Dijkzigt, Rotterdam and to Prof. Dr. H. Esseveld, Head of the Central Bacteriological Laboratory, Rotterdam, for providing facilities for the performance of this study.He thanks Mrs. Dr. H. J. Leijnse-Ybema for her help in making the chromatograms, and Mr. J. B. Lenstra, pharmacist, for advice in matters of organic chemistry.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Novel l-specific cleavage of the urethane bond of t -butoxycarbonylamino acids by whole cells of Corynebacterium aquaticum

Microorganisms capable of cleaving the urethane bond of t-butoxycarbonyl (Boc) amino acids in a whole-cell reaction were screened among stock cultures, and Corynebacterium aquaticum IFO12154 was the most promising. The conversion of Boc-Ala to Ala was stimulated by CoSO₄ in the medium and reaction mixture. The optimum whole-cell concentration was 25 mg lyophilized cells/ml. Boc-l-Met was the best substrate for this reaction, and other Boc-L-amino acids, as well as benzyloxycarbonyl-l-amino acids with hydrophobic residues, were also good substrates. Boc-d- and Z-d-amino acids were inert. When the reactions had proceeded for 24 h with each substrate at 10 mM, the molar conversion rates from Boc-l-, dl- and d-Met were 100%, 50%, and 0% respectively. From 150 mM Boc-l-Met, 143 mM l-Met was formed at a molar yield of 95.3%. Received: 3 September 1996 / Received last revision: 7 April 1997 / Accepted: 19 April 1997     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

D-Amino acids in protein de novo design. II. Protein-diastereomerism versus protein-enantiomerism

Summary With a few exceptions, proteins in our biosphere are based exclusively onl-amino acids. The inversion of configuration of all the stereogenic centers in a protein leads to anall-d compound with ‘mirror image’ properties and ‘mirror image’ structure. We propose to use the termprotein-enantiomerism to describe the relationship between two proteins that have the same sequence but whose amino acids have opposite configuration. We will use the termprotein-diastereomerism to define the relationship between two proteins that have the same sequence in which some amino acids have opposite configurations. A classification of type I, II, III, and IV protein-diastereomerism is proposed. By extension, a diastereoprotein is a protein where some amino acids have the same configuration (l ord) while others have the opposite one (d orl). A particular case of diastereoproteins aremesoproteins, also analyzed in this article. In addition to the goal of making proteins resistant to protease degradation, the use ofd-amino acids in protein de novo design may give rise to proteins with structures, and perhaps properties, very different to those of nativeall-l-proteins.