Fractionation of the enzyme system responsible for gramicidin S biosynthesis

• Published in: Biochimica et biophysica acta Vol. 208; no. 3; pp. 496 - 508
• Main Authors: Otani, Shuzo, Yamanoi, Taketo, Saito, Yoshitaka
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.1970
• Subjects: Adenosine Triphosphate - metabolism; Bacillus - enzymology; Carbon Isotopes; Chemical Precipitation; Chromatography, Gel; Chromatography, Ion Exchange; Enzymes - isolation & purification; Hydroxyapatites; Isomerases; Methods; Phenylalanine; Phosphorus Isotopes; Piperazines; Proline; Quaternary Ammonium Compounds; Sulfates; Tyrothricin - biosynthesis
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(70)90224-2

1. 1. The enzyme system participating in gramicidin S formation was fractionated by precipitation with (NH 4) 2SO 4 and by column chromatography using hydroxylapatite, DEAE-Sephadex amd Sephadex G-200 to investigate the mechanism of gramicidin S biosynthesis. 2. 2. The enzyme system was resolved into four protein fractions, which were designated as Fr-1, Fr-2, Fr-3 and Fr-4 in the order of elution from a hydroxylapatite column. Each separated enzyme fraction failed to form gramicidin S. The formation was achieved when Fr-3 and Fr-4 were combined. It was demonstrated that the addition of Fr-1 to the combined enzyme system was essential in obtaining full enzymatic activity for gramicidin S formation, although the role of Fr-1 in gramicidin S formation was left unclarified. 3. 3. In DEAE-Sephadex column chromatography of Fr-4, a phenylalanine-activating fraction (Fr-4-D-I) was separated from a proline-activating fraction (Fr-4-D-I). With wach separated enzyme fraction, neither gramicidin S nor d-phenylalanyl- L-prolyl diketopiperazine was formed, but D-phenylalanyl- L-prolyl diketopiperazine formation was restored following a combination of these enzyme fractions. When a phenylalanine-activating fraction (Fr-2 + 3-S-II) originating from Fr-2 and Fr-3 was added to Fr-4-D-II, the activity of gramicidin S formation appeared in addition to D-phenylalanyl- L-prolyl diketopiperazine formation. 4. 4. The activating enzymes of the constituent amino acids of gramicidin S except for phenylalanine were not separated by any methods adopted in the experiments.