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Biomedical subjects

H Kleinkauf

Publications and source records attributed to H Kleinkauf.

At least 55 records · Page 3Linked to original sources
PubMed1989

Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154.

The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level beta-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including alpha-aminoadipic acid and cysteine. The Km for alpha-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a beta-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of beta-lactams in bacteria.

Blotting, Western↗
PubMed1989

Proteinchemical and kinetic features of gramicidin S synthetase.

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S.

Amino Acid Isomerases↗
PubMed1989

Cell-free biosynthesis of new cyclosporins.

An enzyme preparation, isolated from extracts of the fungus Beauveria nivea (previously designated Tolypocladium inflatum), is able to synthesize cyclosporins (Cy's) in vitro. At suboptimal temperature it was possible to yield about 50 micrograms of CyA per ml. The enzyme also produces several of the naturally occurring congeners of CyA, such as the Cy's B, C, D, G, M, O, Q, U and V and some of the analogues known to be produced by the fungus via precursor directed biosynthesis, like dihydro-CyA, [N-methyl-L-beta-cyclohexylalanine]CyA, [L-allylglycine]CyA and [D-serine]CyA. Furthermore, Cy's not obtainable by the fungus could be prepared by the enzyme system in the presence of the appropriate precursor amino acids; the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-allo-isoleucine, N-methyl-L-allo-isoleucine]CyA, [L-allo-isoleucine]CyA, [D-2-aminobutyric acid]CyA and [beta-chloro-D-alanine]CyA could be established. The immunosuppressive effects of the new derivatives are discussed.

Chromatography, High Pressure Liquid↗
PubMed1988

Peptide antibiotics, beta-lactams, and related compounds.

In the field of natural peptides, beta-lactams, and related compounds, recent exciting developments are discussed. The increasing interest in this class of bioactive amino-acid derived structures has been attributed to the use of new directed screens (enzyme inhibition assays, beta-lactam detection, immunomodulator studies), new and improved applications (antibiotic, transplantation, and cancer chemotherapy), and advances in functional studies (DNA binding peptides, nucleotide complexones, cell wall and protein processing inhibitors). Peptides offer unique access to modifications and analog production by in vivo (directed biosynthesis) and in vitro procedures (enzymatic synthesis) due to their general linear precursors permitting point replacements. Of special interest are recent developments in the genetics of these compounds (cyclic peptides and beta-lactams), which will find applications in production methods in the near future.

Amino Acid Sequence↗
PubMed1988

Synthesis of beauvericin by a multifunctional enzyme.

Beauvericin synthetase, a multifunctional enzyme catalyzing depsipeptide formation in Beauveria bassiana was purified to near homogeneity. The enzyme consists of a single polypeptide chain with a molecular mass of about 250 kdaltons. The mechanism of beauvericin formation is very similar to that of the cyclohexadepsipeptide enniatin. The constituents of the beauvericin molecule, L-phenylalanine and D-alpha-hydroxyisovaleric acid are activated as thioesters via the corresponding adenylates. N-Methylation takes place at the thioester bound stage of the phenylalanine residues. Omission of the methyl donor S-adenosyl-L-methionine results in the formation of demethylbeauvericin. Studies on substrate specificity revealed that phenylalanine could be replaced by a number of other aromatic or aliphatic amino acids like beta-phenylserine, ortho-, meta-, para-fluorophenylalanine, isoleucine, norleucine and leucine. Valine, the constituent amino acid of enniatin B was not accepted by the enzyme.

Anti-Bacterial Agents↗
PubMed1987

Formation of D-Phe-Pro-Val-cyclo-Orn by gramicidin S synthetase in the absence of L-leucine.

The preparation of both enzymes of gramicidin S synthetase was efficiently improved by introduction of the fast protein liquid chromatography technique. High-resolution anion-exchange chromatography on Pharmacia Mono Q HR 5/5 was used as the final purification step. D-Phe-Pro-Val-cyclo-Orn was obtained as a product of the multienzyme by omission of L-leucine from the complete bioassay mixture. This tetrapeptide was formed by cyclization of the C-terminal ornithine to 3-amino-2-piperidone. It was identified and characterized by chromatographic and spectroscopic procedures using chemically synthesized reference compounds.

Amino Acid Isomerases↗
PubMed1987

Aminoacyl-tRNA synthetases catalyze AMP----ADP----ATP exchange reactions, indicating labile covalent enzyme-amino-acid intermediates.

Aminoacyl-tRNA synthetases (amino acid-tRNA ligases, EC 6.1.1.-) catalyze the aminoacylation of specific amino acids onto their cognate tRNAs with extraordinary accuracy. Recent reports, however, indicate that this class of enzymes may play other roles in cellular metabolism. Several aminoacyl-tRNA synthetases are herein shown to catalyze the AMP----ADP and ADP----ATP exchange reactions (in the absence of tRNAs) by utilizing a transfer of the gamma-phosphate of ATP to reactive AMP and ADP intermediates that are probably the mixed anhydrides of the nucleotide and the corresponding amino acid. AMP and ADP produce active intermediates with amino acids by entering the back-reaction of amino acid activation, reacting with labile covalent amino acid-enzyme intermediates. Gramicidin synthetases 1 and 2, which are known to activate certain amino acids through the formation of intermediate thiol-esters of the amino acids and the enzymes, catalyze the same set of reactions with similar characteristics. Several lines of evidence suggest that these activities are an inherent part of the enzymatic reactions catalyzed by the aminoacyl-tRNA synthetases and gramicidin synthetases and are not due to impurities of adenylate kinase, NDP kinase, or low levels of tRNAs bound to the enzymes. The covalent amino acid-enzyme adducts are likely intermediates in the aminoacylation of their cognate tRNAs. The use of gramicidin synthetases has thus helped to illuminate mechanistic details of amino acid activation catalyzed by the aminoacyl-tRNA synthetases.

Adenine Nucleotides↗
PubMed1987

Peptide uptake by astroglia-rich brain cultures.

Uptake of carnosine has been investigated in astroglia-rich primary cultures derived from brains of newborn mice. It could be demonstrated that carnosine is not degraded by these cells but rapidly taken up in an energy- and sodium-dependent process. Uptake and release of carnosine by these cells were found to be mediated by a saturable, high-affinity transport system with apparent kinetic constants of Km = 50 microM and Vmax = 22.7 nmol X h-1 X mg protein-1. Uptake of carnosine is strongly inhibited by other dipeptides as well as by various oligopeptides, e.g., Leu-enkephalin. However, uptake of the radiolabeled tripeptide D-Ala-L-Ala-L-Ala was not observed. Radiolabeled Leu-enkephalin also did not accumulate intracellularly, even if degradation of the peptide was prevented by use of peptidase inhibitors. These results suggest that uptake of carnosine is catalyzed by a dipeptide-specific transport system with broad substrate specificity. With neuronal cells in primary culture, uptake of carnosine or other peptides was not observed.

Amino Acids↗
PubMed1987

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies.

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.

Affinity Labels↗
PubMed1986

Characterization of two carnosine-degrading enzymes from rat brain. Partial purification and characterization of a carnosinase and a beta-alanyl-arginine hydrolase.

From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes beta Ala-Arg considerably faster than carnosine and, therefore, has been tentatively designated beta Ala-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and beta Ala-Arg (Km = 2 mM). Homocarnosine and gamma-aminobutyryl-arginine are not degraded by beta Ala-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but beta Ala-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, beta Ala-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, beta Ala-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.

Animals↗
PubMed1986

Biosynthesis of cyclosporin A: partial purification and properties of a multifunctional enzyme from Tolypocladium inflatum.

An enzyme fraction most probably involved in the biosynthesis of cyclosporin A was purified 540-fold from Tolypocladium inflatum. The enzyme was capable of forming covalent enzyme-substrate complexes and catalyzed the ATP-pyrophosphate exchange reactions dependent on the unmethylated constituent amino acids of cyclosporin A. Evidence was obtained that covalent binding of substrate amino acids occurred via thioester linkage. Furthermore, the N-methylation of thio-esterified valine, leucine, and glycine residues with S-adenosyl-L-methionine was demonstrated. De novo synthesis of cyclosporin A was not observed but the formation of the diketopiperazine cyclo-(D-Ala-MeLeu) from D-alanine and L-leucine under the consumption of ATP and S-adenosyl-L-methionine. This cyclodipeptide represents a partial sequence of cyclosporin A. Molecular mass determinations revealed the enzyme activity to be lying in the range of about 700 kDa.

Carbon Radioisotopes↗
PubMed1985

Gramicidin S synthetase. Temperature dependence and thermodynamic parameters of substrate amino acid activation reactions.

In the biosynthesis of the cyclic decapeptide antibiotic gramicidin S, the constituent amino acids are activated by a two-step mechanism involving aminoacyl adenylate and thio ester formation which are both reversible processes. The dissociation constants (KD) for the gramicidin S synthetase-substrate amino acid-thio ester complexes are 100-1000-fold lower compared to the KM data of the preceding aminoacyl adenylate reactions. The affinity for these substrates is appreciably higher at the thio template sites than at the aminoacyl adenylate reaction centers. Therefore, the activation equilibria are quantitatively shifted toward thio ester formation. A set of thermodynamic parameters for the activation processes was determined from the temperature dependence of the KM and KD data. Reaction enthalpies were obtained from a van't Hoff analysis of these constants. delta G degree for the substrate activation reactions of the heavy enzyme of gramicidin S synthetase (GS 2) is predominantly controlled by entropy contributions. In contrast, the overall activation and concomitant racemization of phenylalanine by phenylalanine racemase (GS 1) are exothermic processes which are distinguished by a small negative reaction entropy.

Amino Acid Isomerases↗
PubMed1985

Molecular cloning of an ornithine-activating fragment of the gramicidin S synthetase 2 gene from Bacillus brevis and its expression in Escherichia coli.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.

Amino Acid Isomerases↗
PubMed1985

Reactive sulfhydryl groups involved in the aminoacyl adenylate activation reactions of the gramicidin S synthetase 2.

Six accessible sulfhydryl groups of the light component of gramicidin S synthetase (GS 1) were titrated with N-ethylmaleimide (Ma1NEt) as well as 3,3'-dithiobis(6-nitrobenzoic acid) (Nbs2). Twenty-four thiols were detected in the heavy enzyme (GS 2) using Ma1NEt as the modifier. Substrate amino acid-induced protection of GS 2 against deactivation by Ma1NEt indicates that in addition to the specific thiols at the thiotemplates of gramicidin S synthetase reactive SH groups are also involved in the primary aminoacyl adenylate activation reactions. Obviously 2 sulfhydryl groups are essential for the adenylation of each L-Pro, L-Val and L-Leu, while 3 thiols were detected for the ornithine activation. Agents like Ma1NEt or Nbs2 inhibit the thiolation of the substrate amino acids of gramicidin S synthetase and gramicidin S formation more severely than the aminoacyl adenylate activation reactions which are affected at 10-100-fold higher inhibitor concentrations. These processes can be studied separately if the thioester formation sites are inhibited at low concentrations of sulfhydryl inhibitors.

Adenosine Triphosphate↗
PubMed1984

4-Methyl-3-hydroxyanthranilic acid activating enzyme from actinomycin-producing Streptomyces chrysomallus.

A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.

Adenosine Triphosphate↗
PubMed1984

D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea.

A D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea was purified about 145-fold. The enzyme was able to catalyse both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Both reactions were also catalysed to a decreased but significant extent with respect to dihydrolysergic acid. The molecular mass of the enzyme was estimated to lie between 135 and 140 kDa. The involvement of the enzyme in the biosynthesis of ergot peptide alkaloids is discussed.

Adenosine Triphosphate↗
PubMed1984

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea.

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca or Mg, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.

Journal Article↗