scholarly journals Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas

2005 ◽  
Vol 390 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Jing Wu ◽  
Galina Ya. Sheflyan ◽  
Ronald W. Woodard
Keyword(s):  
Bacillus Subtilis ◽  
Dipicolinic Acid ◽  
Shikimate Pathway ◽  
Chorismate Mutase ◽  
Highly Active ◽  
Bifunctional Enzymes ◽  
Iron And Zinc ◽  
Bivalent Metal Ions ◽  
Structure In Solution ◽  
Phosphate Synthase

The mono/bifunctional and metallo/non-metallo properties of Bacillus subtilis DAHPS (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) have been controversial for several decades. The present study investigated the DAHPSs from both the B. subtilis parent Marburg strain and the derivative strain 168 in detail and clarified the above two long-standing questions. The DAHPSs from the parent and the derivative 168 strains have identical sequence and are both bifunctional enzymes with a CM (chorismate mutase) activity and a DAHPS activity. The parent strain expresses a second independent monofunctional CM, encoded by aroH, that is highly active, while the 168 strain expresses an aroH containing a single residue mutation (A112V) that is significantly less active thus leading to previous confusion regarding the mono/bifunctionality of DAHPS. Metal analysis showed that B. subtilis DAHPS as isolated contained iron and zinc and is inactivated by dipicolinic acid; the inactive apoenzyme can be reactivated by bivalent metal ions, indicating that the enzyme is a metalloenzyme. The enzyme-bound metal is insensitive to EDTA treatment, leading to the previous conclusion that this DAHPS does not require a metal. The enzyme displays a homotetrameric structure in solution and appears to follow Michaelis–Menten kinetics with KmPEP=139±11.4 μM for phosphoenolpyruvate, KmE4P=1760±110 μM for D-erythrose 4-phosphate, kcat=4.6±0.1 s−1 for DAHPS activity and Kmchorismate=850±97 μM, kcat=0.41±0.01 s−1 for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.

The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis

1981 ◽  
Vol 1 (5) ◽  
pp. 426-438
Author(s):  
G S Byng ◽  
R J Whitaker ◽  
C L Shapiro ◽  
R A Jensen
Keyword(s):  
Amino Acid ◽  
Euglena Gracilis ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Shikimate Pathway ◽  
Exchange Chromatography ◽  
Chorismate Mutase ◽  
Nicotinamide Adenine ◽  
Acid Pathway ◽  
Phosphate Synthase

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.

scholarly journals The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis.

1981 ◽  
Vol 1 (5) ◽  
pp. 426-438 ◽  
Author(s):  
G S Byng ◽  
R J Whitaker ◽  
C L Shapiro ◽  
R A Jensen
Keyword(s):  
Amino Acid ◽  
Euglena Gracilis ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Shikimate Pathway ◽  
Exchange Chromatography ◽  
Chorismate Mutase ◽  
Nicotinamide Adenine ◽  
Acid Pathway ◽  
Phosphate Synthase

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.

scholarly journals Molecular Physiological Characterization of a High Heat Resistant Spore Forming Bacillus subtilis Food Isolate

2021 ◽  
Vol 9 (3) ◽  
pp. 667
Author(s):  
Zhiwei Tu ◽  
Peter Setlow ◽  
Stanley Brul ◽  
Gertjan Kramer
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Laboratory Strain ◽  
High Heat ◽  
High Heat Resistance ◽  
Vegetative Cells ◽  
Heat Resistant ◽  
Food Isolate ◽  
Wet Heat

Bacterial endospores (spores) are among the most resistant living forms on earth. Spores of Bacillus subtilis A163 show extremely high resistance to wet heat compared to spores of laboratory strains. In this study, we found that spores of B. subtilis A163 were indeed very wet heat resistant and released dipicolinic acid (DPA) very slowly during heat treatment. We also determined the proteome of vegetative cells and spores of B. subtilis A163 and the differences in these proteomes from those of the laboratory strain PY79, spores of which are much less heat resistant. This proteomic characterization identified 2011 proteins in spores and 1901 proteins in vegetative cells of B. subtilis A163. Surprisingly, spore morphogenic protein SpoVM had no homologs in B. subtilis A163. Comparing protein expression between these two strains uncovered 108 proteins that were differentially present in spores and 93 proteins differentially present in cells. In addition, five of the seven proteins on an operon in strain A163, which is thought to be primarily responsible for this strain’s spores high heat resistance, were also identified. These findings reveal proteomic differences of the two strains exhibiting different resistance to heat and form a basis for further mechanistic analysis of the high heat resistance of B. subtilis A163 spores.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

Effects of Cold Atmospheric Pressure Plasma Jeton the Viability of Bacillus subtilis Endospores

2013 ◽  
Vol 647 ◽  
pp. 524-531
Author(s):  
Vinita Sharma ◽  
Katsuhiko Hosoi ◽  
Tamio Mori ◽  
Shin-ichi Kuroda
Keyword(s):  
Bacillus Subtilis ◽  
Atmospheric Pressure ◽  
Dipicolinic Acid ◽  
Atmospheric Pressure Plasma ◽  
Dilution Method ◽  
Pressure Plasma ◽  
Agar Disc ◽  
Bacterial Endospores ◽  
Cold Atmospheric Pressure Plasma ◽  
Ar Gas

In this study, we conducted experiments to investigate the effectiveness of a non-equilibrium Ar-N2 plasma jet generated by a Cold Atmospheric Pressure Plasma Torch (CAPPLAT) at a sinusoidal voltage of 20 kV, frequency of 30 kHz with 10 slm of Ar gas and 100 sccm of N2 gas. Highly environmental stress resistant bacterial endospores of Bacillus subtilis, dried on an agar disc were exposed to the plasma discharge from the CAPPLAT for different durations. The viability of spores after plasma exposure was checked by counting CFUs by serial dilution method. We also measured the amount of released DPA (dipicolinic acid, pyridine-2, 6-dicarboxylic acid), which is exclusively found in endospore protoplast (cortex), to confirm the disintegration of the cortex. We could successfully inactivate a population of Bacillus endospores of about 1.0 × 107 to 4.0 × 107 spores/ml.

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