Biomedical subjects
Publications and source records attributed to V Bĕhal.
Hybrid antibiotics that do not occur in nature have been obtained by combining structural genes of antibiotic producers. Some of these substances were effective against pathogenic microorganisms resistant against antibiotics produced by the parent strains. The majority of hybrid antibiotics were obtained by combining genes encoding polyketide synthases. Hybrid peptides with new biological properties have also been synthesized.
The chapter informs about different types of antibiotics, their structure, biosynthesis and their regulation. Industrial cultivation and isolation of antibiotics is described in the chapter. Search for microorganisms producing antibiotics and preparation of high-producing strains is described. Resistance against antibiotics in producing microorganisms and pathogens is discussed.
Microorganisms produce low-molar-mass secondary metabolites exhibiting different biological activities, which are used. e.g., in medicine as antimicrobial and antifungal agents, alkaloids and toxins. Some of these substances have highly diverse biological activities and unusual structures. They are produced by streptomycetes, fungi, and bacilli, but interesting products have also been obtained from microorganisms growing in extreme conditions. Several thousands of microbial products have so far been discovered and many other, which can be potentially useful and/or prospective for human use, can still be in the offing.
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Glutamate was converted to glutamine by Corynebacterium glutamicum permeabilized by ethanol (10%). ATP was supplied to the reaction by dried Saccharomyces cerevisiae or regenerated by acetyl kinase. High glutamine synthetase activity in C. glutamicum was induced by cultivation of the microorganism in media containing glutamate as the sole source of nitrogen.
The first valine dehydrogenase of S. aureofaciens had been described (Vancurová, I., Vancura, A., Volc, J., Neuzil, J., Flieger, M., Basarová, G. and Bĕhal, V. (1988) J. Bacteriol. 170, 5192-5196). In the present work, a second valine dehydrogenase was detected and purified by hydrophobic and fast protein liquid chromatographies. The enzyme has a relative molecular mass (M(r)) of 240,000 and is composed of 6 identical subunits, each of M(r) 41,000. In the presence of NAD, the enzyme catalyzes the reversible deamination of several branched- and straight-chain amino acids. The enzyme activities with L-2-aminobutyrate and deamino-NAD+ are markedly higher than those with L-valine and NAD+, respectively. The enzyme synthesis is significantly induced by L-valine but severely repressed by ammonia. Molecular and catalytic properties of the enzyme distinguish it from the other described valine dehydrogenases. The results directly demonstrate the presence of two valine dehydrogenases in a single Streptomyces species.
Sterol synthesis in Saccharomyces cerevisiae was primarily controlled by the growth rate. At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells. At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2-2.5%, which seems to be the maximum value for S. cerevisiae. After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols. The sporulating S. cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter.
A new micromethod for measuring enzyme-catalyzed reactions was developed. The method involves a number of consecutive direct injections of aliquots of the reaction mixture onto a microbore column and permits the determination of the time dependence of the decrease of substrate or increase of product concentrations. The reactions proceed in a microvial placed in the autosampler, and as the starting volume can be as low as 10 microliters, the requirement for the amount of enzyme is very low. The autosampler backed by the liquid chromatographic software allows automation of the analyses including data processing and easy quantitation of the enzymatic reaction(s). The method was applied to a system of two consecutive terminal reactions of tetracycline biosynthesis in Streptomyces aureofaciens, catalyzed by anhydrotetracycline oxygenase and tetracycline dehydrogenase. The usage of a diode-array detector facilitated the quantification of the reactions as the product and substrate could be monitored at their optimal wavelengths.
Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.
Homogeneous alanine dehydrogenase isolated from Streptomyces aureofaciens, a producer of tetracycline, was characterized from the point of its molecular and catalytic properties. Using analytical ultracentrifugation the molecular weight of alanine dehydrogenase was found to be 198,000. The enzyme could use as cofactors apart from NAD+ also 1,N6-etheno-NAD+, 3-acetylpyridine-NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide. The enzyme activity in the direction of oxidative deamination was not affected by the addition of nonsubstrate amino acids, however, it was sensitive to inhibitors of SH-groups. Reductive amination of pyruvate was inhibited by L-alanine, L-serine and D-alanine. The inhibition by L-alanine and L-serine was uncompetitive with respect to NADH and noncompetitive with regard to pyruvate and ammonium ions.
Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.
Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.
Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.
Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.
Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.
Anhydrotetracycline oxygenase was purified both by affinity chromatography and by hydrophobic interaction chromatography. Molecular weight of anhydrotetracycline oxygenase was determined to be 115,000 by Sephadex G-200 gel filtration. Using preparative isoelectric focusing the isoelectric point of the enzyme was estimated to be 5.3. The enzyme showed a sensitivity to thiol-specific inhibitors. During the hydrophobic interaction purification step, the activity dropped considerably. Reactivation occurred when a heat treated crude extract was added to the reaction mixture.
Mycelia of a low- and a high-production strain of Streptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2). The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 microM) was present in the growth medium.