Lieu Thi Nguyen

The tylosin producer, Streptomyces fradiae, contains a second valine dehydrogenase

A second NAD-dependent valine dehydrogenase (VDH) of Streptomyces fradiae was detected and purified to homogeneity by affinity chromatography on Reactive-Blue 2 Sepharose followed by gel filtration and Mono Q fast protein liquid chromatography. The relative molecular masses of the native enzyme and its subunits were determined to be 80,000 and 41,000, respectively, indicating that the enzyme is a homodimer. The enzyme was the only active VDH in S. fradiae; its activity was significantly induced by L-valine, but was repressed by ammonia. Among branched- and straight-chain amino acids that serve as enzyme substrates, L-2-aminobutyrate and L-valine are preferred. Significant activities were found with deamino-NAD+ and 3-pyridinealdehyde-NAD+. The molecular and catalytic properties of the enzyme distinguish it from the enzyme previously purified, and thus indirectly indicate the existence of two VDHs in S. fradiae.

Purification and characterization of a novel valine dehydrogenase from Streptomyces aureofaciens

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.

What type of glutamine synthetase is important forStreptomyces coelicolor A3(2) under nitrogen-limited growth conditions?

SummaryGlutamine synthetase I activity ofStreptomyces coelicolor was strongly repressed by ammonia and was induced 56.8 fold in a nitrogen-free medium. Glutamine synthetase II activity was not induced even by a long-term nitrogen starvation. Therefore, glutamine synthetase I is the only active enzyme ofStreptomyces coelicolor.

The induction of valine dehydrogenase activity from Streptomyces by L-valine is not repressed by ammonium

SummaryActivity of valine dehydrogenases (VDH) from Streptomyces aureofaciens and S. fradiae is strongly induced by L-valine even in the presence of 25mM NH4+. When added into 16 h-old cultures growing with 100mM NH4+, L-valine induced the synthesis of VDH. The results indicate that Streptomyces can utilize L-valine in the presence of NH4+, and the induction of VDH activity by L-valine is not repressed by NH4+.

DoesStreptomyces aureofaciens contain two alanine dehydrogenase isozymes

SummaryTwo NAD-dependent dehydrogenases exhibiting activity with L-alanine were detected in crude extracts ofStreptomyces aureofaciens. The dominant enzyme was found to be an alanine dehydrogenase with 6 or 4 subunits, each ofM, 45,000. The minor enzyme exhibited high activities with branched-chain amino acids, suggesting it is a valine dehydrogenase. The results demonstrate thatS. aureofaciens has only one alanine dehydrogenase, which is catalytically active in a six- or four-subunit structure.

How is glutamine synthetase I activity from Streptomyces aureofaciens regulated

SummaryActivity of glutamine synthetase I (GSI) from Streptomyces aureofaciens increased markedly during tetracycline production phase. The purified GSI exhibited a low affinity for glutamate but high affinities for ATP and ammonium. Its Mn2+-dependent activity was more sensitive to feedback inhibitors than Mg2+-dependent activity. Both the activities were significantly stimulated by Co2+ but inhibited by other divalent cations. ADP was a strong inhibitor. These results suggest that GSI activity is regulated by availability of substrates, feedback inhibitors, divalent cations, and cell energy charge.

Identification and characterization of a heat-labile type I glutamine synthetase fromStreptomyces cinnamonensis

Streptomycetes have two distinct glutamine synthetases (GS): a heat-stable dodecameric GSI and a heat-labile octameric GSII. A heat-inactivated GS activity was detected in crude extracts ofStreptomyces cinnamonensis cells grown with nitrate or glutamate as the nitrogen source. The purified enzyme obtained from crude extracts of the nitrate-grown cells after affinity and anion-exchange chromatography was also heat-labile; it was inactivated by 80 % when incubated at 50 °C for 1 h. However, the enzyme has properties typical of GSI and similar with those of the heat-stable GSI purified fromS. aureofaciens: It is composed of twelve subunits, each ofM 55 kDa, and has a native molar mass of 625 kDa and an isoelectric point at pH 4.2. In addition, its activity is regulated by reversible adenylylation. Mg2+ and NaCl but not Mn2+ protected the purified enzyme from thermal inactivation, and both NaCl and Mn2+ or Mg2+ stabilized its activity at 4–8 °C. As compared with GSI fromS. aureofaciens, theS. cinnamonensis enzyme was cleaved more extensively during SDS-PAGE, was less sensitive to feedback inhibitors, and similarly affected by divalent cations. TheKm values were 12.5 mmol/L forl-glutamate, 0.1 for NH4+, 1.25 for ATP, 18.5 forl-glutamine, 3.3 for hydroxylamine and 0.087 for ADP. To our best knowledge, this is the first report of a heatlabile GSI from any source.

Removal of Mn2+ induces dissociation of glutamine synthetase fromStreptomyces aureofaciens

Removal of Mn2+ by EDTA treatment converted dodecameric glutamine synthetase (GS) fromStreptomyces aureofaciens into inactive subunits but did not affect significantly their conformation. However, when fractionated by gel filtration FPLC, the Mn2+-free subunits showed a 7-fold increase ofA280, probably due to a significant alteration in their tertiary structure. Mn2+ reduced theA280 of the subunits and promoted their reaggregation to form active GS. Mg2+ or Ca2+ but not Co2+ or Zn2+ might have similar effects. The results suggest that specific divalent cations might play a crucial role in stabilizing subunit interactions as well as the conformation of the individual subunits inStreptomyces GS. The role of specific divalent cations in the regulation of GS turnover is discussed.

Regulation, purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg2+ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn2+ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.

The avermectin-producing Streptomyces avermitilis possesses an inducible valine dehydrogenase

SummaryValine dehydrogenase (VDH) is believed to be absent in Streptomyces avermitilis. In the present study, a VDH (Mr, 72 000) was detected by activity measurement and activity staining on a native-PAGE gel. The enzyme activity was induced by L-valine and repressed by ammonia. VDH activity was found to be significantly lower than L-valine transaminase activity. The results suggest that one active VDH does exist in S. avermitilis, and plays a role in valine catabolism and avermectin biosynthesis.