Screening, overexpression and characterization of an N-acylamino acid racemase from Amycolatopsis orientalis subsp. lurida

Thirty-one different actinomycete strains were used in a genetic screening using PCR and Southern hybridization methods to detect N-acetylamino acid racemases (AAR) in order to obtain enzymes with different properties. Cloning and sequencing of a 2.5 kb EcoRI DNA fragment from Amycolatopsis orientalis subsp. lurida revealed the coding gene of an N-acetylamino acid racemase, which had identities to the aar gene of Amycolatopsis sp. TS-1-60 [Tokuyama and Hatano (1995) Appl Microbiol Biotechnol 42:884-889] of 86% at the level of DNA, and 90% at the level of amino acids. The heterologous overexpression in Escherichia coli resulted in a specific activity of about 0.2 U/mg of this racemase. A two-step purification with heat treatment followed by anion-exchange chromatography led to almost homogeneous enzyme. The optimum pH of the enzyme was 8.0 and it was stable at 50 degrees C for 30 min. The relative molecular mass of the native enzyme and the subunit was calculated to be 300 kDa and 40 kDa by gel filtration and SDS-PAGE, respectively. The isoelectric point (pI) of the AAR was 4.4. It catalyzed the racemization of optically active N-acetylamino acids such as N-acetyl-L- or -D-methionine and N-acetyl-L-phenylalanine. Further characterization of the racemase demonstrated a requirement for divalent metal ions (Co2+, Mn2+, Mg2+) for activity and inhibition by EDTA and p-hydroxymercuribenzoic acid. AAR is sensitive to substrate inhibition at concentrations exceeding 200 mM.     

Biochemical composition of washed human seminal coagulum in comparison to sperm-free semen from the same donors

Intra-individual inter-ejaculate variations in the amounts of protein, fructose, N-acetylamino sugar, phosphate, sialic acid and amino sugar in washed coagulum from normal ejaculates of men were highly consistent (N = 9). All the prostatic components studied (acid phosphatase, zinc, calcium and citric acid), except zinc, in the washed coagulum were reduced by 90% of their values in semen (N = 5). The seminal vesicular markers (fructose, N-acetylamino sugar and phosphate) had no association with the coagulum structure and represented the soluble components (N = 5). The concentrations of protein and zinc in the coagulum were higher than those of semen by 114% and 32% respectively. The coagulum contained sialic acid and amino sugar as integrated components.     

N-acetylamino acid utilization by kidney aminoacylase-1

Mammalian aminoacylase-1 (Acy1) participates in the breakdown of N-acetylated amino acids during intracellular protein catabolism. Acy1 is most abundantly expressed in the kidney tubular epithelium. Lately, Acy1 deficiency was identified in children with increased urinary excretion of several N-acetylamino acids. Here we report detailed N-acetylamino acid specificity profiles for human and porcine Acy1 based on steady state kinetic measurements. We found that LLC-PK1 cells, a model of the porcine kidney proximal tubular epithelium, robustly express Acy1. For the first time, we demonstrate uptake and utilization of N-acteylleucine and -methionine in replacement of the free amino acid, respectively, in cultured epithelial cells. Our data are consistent with a specific role of kidney Acy1 in the salvage of amino acids originating from systemic degradation of N-acetylated proteins.     

Hydrophobic benzoic acids as inhibitors of influenza neuraminidase

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A new benzoic acid inhibitor (11) containing a lipophilic side chain at C-3 and a guanidine at C-5 was synthesized. The X-ray structure of 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid in complex with NA revealed that the lipophilic side chain binds in a newly created hydrophobic pocket formed by the movement of Glu 278 to interact with Arg 226, whereas the guanidine of 11 interacts in a negatively charged pocket created by Asp 152, Glu 120 and Glu 229. Compound 11 was highly selective for type A (H2N2) influenza NA (IC50 1 microM) over type B (B/Lee/40) influenza NA (IC50 500 microM).     

Rearrangement of 3-ketoceramide.

N-Acetyl 3-ketosphingosine was converted under mild alkaline conditions to an unknown nonpolar compound with a good yield. From its mass, infrared, and NMR spectra, the structure of the unknown compound was deduced to be 2-tridecyl-5-(N-acetylamino)-tetrahydro-γ-pyrone. This rearrangement occurred by an intramolecular Michael-type addition reaction.     

Some amino sugars structurally related to 6-deoxymannojirimycin precursors prepared from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose and methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside

Methyl (5S)-5-C-amino-5-cyano-5-deoxy-2,3-O-isopropylidene-alpha-D-lyxofuranoside has been synthesised from methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside, applying the Strecker synthesis. Analogously, methyl (5S) and (5R)-5-C-amino-5-cyano-5,6-dideoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosides were prepared from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose. The 5-S configuration was unambiguously determined by single-crystal X-ray diffraction analysis of corresponding N-acetyl derivatives. Conformations of five-membered rings are discussed. The conversion of N-acetylated amino nitriles to N-acetylamino acid ethyl ester and amide, respectively, is also described.     

Substrate specificity of acetoxy derivatives of coumarins and quinolones towards Calreticulin mediated transacetylation: investigations on antiplatelet function

Calreticulin transacetylase (CRTAase) is known to catalyze the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP), thus modulating their activity. Herein, we studied for the first time the substrate specificity of CRTAase towards N-acetylamino derivatives of coumarins and quinolones. This study is endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. Among all the N-acetylamino/acetoxy coumarins and quinolones screened, 7-N-acetylamino-4-methylcoumarin (7-AAMC, 17) was found to be the superior substrate to platelet CRTAase and emerged as the most promising antiplatelet agent both in vitro and in vivo. Further it caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2), modulation of tissue factor and the inhibition of platelet aggregation. It was also found effective in the inhibition of LPS induced pro-thrombotic conditions.     

Determination of amino sugars and amino acids in glycoconjugates using precolumn derivatization with o-phthalaldehyde.

This report examines the RP-HPLC separation of o-phthalaldehyde derivatives of amino acids, amino sugars, and amino sugar alcohols using either 2-mercaptoethanol or 3-mercaptopropionic acid. A method with pmol sensitivity for the analysis of N-acetylamino sugars of glycoconjugates was elaborated. Upon hydrolysis, amino sugars are reduced with borohydride. Automated precolumn derivatization and chromatographic conditions for the resulting hexosaminitols are the same as those used for the analysis of amino acids. The method has been tested with as little as 2 micrograms of bovine fetuin, with a glycopeptide from bromelain and with an oligosaccharide after periodate oxidation.     

The mean conformation of N-acetylamino acid N'-methylamides in dimethyl sulfoxide solution

The mean conformation of a series of N-acetylamino acid N'-methylamides in dimethyl sulfoxide (DMSO) was determined by 1H-NMR and 13C-NMR methods.The series investigated consisted of derivatives of DL-Ala, DL-Asn, Gln, His, DL-Ile, DL-Leu, Met, DL-Pro, DL-Phe, Ser, Thr, Trp, Tyr, DL-Val, DL-Nva, and dl-Nle. It was found that the conformational equilibria in DMSO are dominated by C5 and C7eq forms.The amounts of these forms in the equibbria were found to be roughly proportional to the Boltzmann probabilities for the occurrence of a definite form, as calculated theoretically by Vasquez et al.(Macromolecules 16 (1983) 1043).Exceptions to this rule were DL-Pro and, to a lesser extent, Ser, Asn and Trp derivatives.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-D-glucosamine.

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-D-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-D-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

Isolation procedure and properties of monomer unit from lysozyme digest of peptidoglycan complex excreted into the medium by penicillin-treated Brevibacterium divaricatum mutant.

1. The peptidoglycan complex excreted in large amounts into the medium by the biotin-requiring mutant Brevibacterium divaricatum NRRL-2311 incubated in the presence of penicillin for 1 h has been investigated. A convenient isolation procedure with high yield for the pure monomeric unit from lysozyme digest of the accumulated polymer is described. 2. It is shown that the released peptidoglycan possesses the linear uncross-linked structure made of repeating disaccharide-pentapeptide unit [GlcNAc-MurNac-Ala-D-Glyn(meso-DAP-D-Ala-D-Ala)] which was isolated by stepwise gel filtration and fractionation of the digestion mixture in 10-mg quantities. Evidence that the minor digestion product of accumulated peptidoglycan possesses the glycan-linked dimer structure is given. Under conditions of beta-elimination, the monomeric unit yielded a lactylpentapeptide which was isolated in pure form by gel filtration. 3. The monomer unit originating from the cultures to which L-[U-14C]glutamic acid was added simultaneously with penicillin incorporated the label exclusively in the peptide chain, whereas that labeled from E11-14C]acetate as the precursor contained radioactivity in both the peptide chain (53%) and N-acetylamino groups (47%) of the glycan portion.     

Comparative rates of esterification of 5′-AMP with hydrophobic amino acids: Relevance to the genetic-code assignments

We have continued our program aimed at understanding the origin and evolution of the genetic code and the process of protein synthesis by comparing the rates of esterification of 5'-AMP by a series of hydrophobic N-acetylamino acids. The reaction clearly shows differences in reaction rate (AcPhe greater than AcLeu greater than AcVal greater than AcIle) among the amino acids having A as middle letter of their anticodons. However, there were no significant differences in reaction rate between AcLeu, AcNorleu, and Ac-alpha-aminobutyric acid, and AcGly reacted faster than all of these and AcPhe. Consequently, this simple reaction with AMP can distinguish only among those amino acids that actually have A as the middle anticodonic nucleotide. The relevance of these studies to the origins of the process of protein synthesis and of the genetic code is discussed in conjunction with results from other studies of a similar nature.     

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.     

Enhancement of bioactivity of Saccharomyces cerevisiae alpha-mating factor by attachment of sugar moiety to glutamine residue

We prepared yeast Saccharomyces cerevisiae alpha-mating factor, a 13-amino acid pheromone produced by haploid alpha-cells, bound with glucose or N-acetylglucosamine at the fifth glutamine residue from the N-terminal by the chemical method of peptide synthesis. It was found that the bioactivity of glucosyl alpha-mating factor was higher than that of native alpha-mating factor. However, it was slightly lower than that of N-acetylglucosaminyl alpha-mating factor. This suggested that the N-acetylamino residue might play some important role in the enhancement of the bioactivity of alpha-mating factor. However, CD spectra analysis of alpha-mating factor and its derivatives demonstrated that their structures were almost identical. On the other hand, we attached a sialo complex type oligosaccharide to N-acetylglucosamine or its glucose residue by means of the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). The attachment of the oligosaccharide to both alpha-mating factors reduced their activities. However, enzymatical trimming of the sialo complex type oligosaccharide recovered its activity.     

A reaction for the simple sensitive fluorimetric assay of heparin and 2-amino sugars

1. 3,5-Diaminobenzoic acid reacted rapidly with the product from HNO(2) deamination of heparin, heparan sulphate and 2-amino-2-deoxyhexoses under very mild conditions (pH3.0 and 37 degrees C) to give stable fluorescent derivatives. 2. The fluorescence yield was rectilinearly related to the concentration of heparin etc. Less than 0.1mug of 2-amino-2-deoxyhexose was easily measurable in standard cuvettes. 3. The deamination products of glucosamine and (particularly) galactosamine were labile in the HNO(2) reagent, with half-lives of 20-40min at room temperature. At 0 degrees C they were much more stable. The analogous product from heparin was not so labile. 4. Under the standard conditions, and at room temperature, relative fluorescence yields (d-glucosamine=1.0) were: d-galactosamine, 0.75; d-gulosamine, 0.38; d-mannosamine, approx. 0.20. 5. Neutral sugars, chondroitin sulphates, DNA and N-acetylneuraminic acids did not react, nor did N-acetylamino sugars or non-deaminated hexosamines. 6. It is suggested that the Dische-Borenfreund [Dische & Borenfreund (1950) J. Biol. Chem.184, 517-522] indole method, the Kissane-Robins [Kissane & Robins (1962) J. Biol. Chem.233, 184-188] DNA assay and the proposed amino sugar method are all examples of simple aldehyde reactions. The specificity of the proposed method is considerably greater than that of the Dische-Borenfreund procedure, partly because of the much milder reaction conditions. 7. The proposed method is very reproducible, about 50-100 times as sensitive as the Elson-Morgan reaction, and 10-50 times as sensitive as the Dische-Borenfreund procedures. It is also convenient; acid hydrolysates of amino sugar-containing compounds can be directly neutralized with sodium acetate solution.     

Synergistic effects in the designs of neuraminidase ligands: Analysis from docking and molecular dynamics studies

Docking and molecular dynamics were used to study the nine ligands (see Scheme 1) at the neuraminidase (NA) active sites. Their binding modes are structurally and energetically different, with details given in the text. Compared with 1A (oseltamivir carboxylate), the changes of core template or/and functional groups in the other ligands cause the reductions of interaction energies and numbers of H-bonds with the NA proteins. Nonetheless, all these ligands occupy the proximity space at the NA active sites and share some commonness in their binding modes. The fragment approach was then used to analyze and understand the binding specificities of the nine ligands. The contributions of each core template and functional group were evaluated. It was found that the core templates rather than functional groups play a larger role during the binding processes; in addition, the binding qualities are determined by the synergistic effects of the core templates and functional groups. Among the nine ligands, 1A (oseltamivir carboxylate) has the largest synergistic energy and its functional groups fit perfectly with the NA active site, consistent with the largest interaction energy, numerous H-bonds with the NA active-site residues as well as experimentally lowest IC₅₀ value. Owing to the poorer metabolizability than oseltamivir, large contribution of the benzene core template and fine synergistic effects of the functional groups, the 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid should be an ideal lead compound for optimizing NA drugs.     

Acyl-peptide hydrolase from rat liver. Characterization of enzyme reaction

Acyl-peptide hydrolase, which catalyzes the hydrolysis of an N-terminally acetylated peptide to release an N-acetylamino acid, was isolated from rat liver and found to be N-terminally blocked. The kinetics of the hydrolysis of acetyl (Ac)-Ala-Ala, Ac-Ala-Ala-Ala, acetylalanine p-nitroanilide, and acetylalanine beta-naphthylamide were investigated. The Km values were between 1 and 9 mM, and the Vmax values were between 100 and 500 nmol/min/micrograms of enzyme. The enzyme activity toward acetylalanine p-nitroanilide and acetylalanine beta-naphthylamide was activated by the presence of Cl- and SCN- at concentrations between 0.1 and 0.5 M. By contrast, the activity toward Ac-Ala-Ala and Ac-Ala-Ala-Ala was inhibited by these anions. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by the addition of diisopropyl fluorophosphate, diethyl pyrocarbonate. Woodward's Reagent K, and glycine methyl ester/carbodiimide. Titration by diisopropyl fluorophosphate showed 0.7 mol of active serine/mol of enzyme subunit, which was confirmed by the incorporation of [3H]diisopropyl fluorophosphate into the enzyme. Acetylalanine chloromethyl ketone inactivated the enzyme following pseudo-first order kinetics; and Ac-Ala, a competitive inhibitor, protected the enzyme from this inactivation. Acyl-peptide hydrolase appears to be a serine protease utilizing a charge relay system involving serine, histidine, and, probably, a carboxyl group(s). Two series of acetyl dipeptides, acetylamino acid p-nitroanilides and acetylamino acid beta-naphthylamides, were prepared in order to determine enzyme specificity. The enzyme preferentially removed Ac-Ala, Ac-Met, and Ac-Ser, the most common acetylated N-terminal residues (Persson, B., Flinta, C., von Heijne, G., and J?rnvall, H. (1985) Eur. J. Biochem. 152, 523-527). The enzyme was shown to be useful for deblocking peptides (e.g. alpha-melanocyte-stimulating hormone and acetyl-renin substrate), and the crude enzyme/substrate mixtures were amenable to direct protein sequence analysis.     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Differential activation and inhibition of lymphocyte proliferation by modulators of protein kinase C: diacylglycerols, "rationally designed" activators and inhibitors of protein kinase C

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) can enhance or inhibit lymphocyte proliferation. Enhancement correlated with increased interleukin 2 (IL-2) production and activation of protein kinase C while inhibition correlated with decreased IL-2 and downregulation of protein kinase C activity (D.S. Grove and A.M. Mastro, Cancer Res. 51, 82-88). In this study, various activators and inhibitors of protein kinase C were used in order to try to separate the effects of TPA on this enzyme from its effects on IL-2 production and determine if protein kinase C activity was directly or indirectly related to IL-2 production. 1,2-Dioctanoylglycerol, 1-oleoyl-2-acetyl-glycerol, phospholipase C, and two "rationally designed" activators, 6-(N-decylamino)-4-hydroxy-methylindole and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, were tested. Some activators enhanced proliferation in the presence of a Ca2+ ionophore, ionomycin, but not concanavalin A. Some activators suppressed proliferation and downregulated protein kinase C. Others neither downregulated protein kinase C nor inhibited IL-2 production and proliferation. However, inhibition or downregulation of protein kinase C activity always correlated with decreased IL-2 and depressed proliferation. Thus, the evidence in this and the previous study suggests that activation of protein kinase C is directly related to IL-2 production in activated T cells.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.