Ivana Vančurová

Valine Dehydrogenase from Streptomyces fradiae: Purification and Properties

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+.

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. KM values were 5.0 mM for L-alanine and 0.11 mM for NAD+. KM values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH4Cl.

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae

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.

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae, a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in α-aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in α-aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.

A direct-injection reversed-phase liquid chromatographic micromethod for studying the kinetics of terminal reactions of tetracycline biosynthesis

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.

High-Performance Liquid Chromatography of Derivatives Involved in the Terminal Steps of Tetracycline Biosynthesis in Streptomyces Aureofaciens

Abstract A simple and reproducible method was developed for the analysis of tetracycline derivatives involved in the last two steps of tetracycline biosynthetic pathway in Streptomyces aureofaciens. The method is based on a gradient liquid chromatographic separation of the compounds using a microbore octadecyl silica column. Beside separation of a mixture of standards, the method was used for separation, detection and quantitation of dehydrotetracycline and tetracycline prepared enzymatically in vitro using anhydrotetracycline oxygenase and tetracycline dehydrogenase, respectively, isolated from S. aureofaciens. The method permits a simple and accurate characterization of kinetics of corresponding enzymatic activities.