Functional characterization of constituent enzyme fractions of mycobacillin synthetase

Abstract

The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.

Role of ATP and enzyme-bound nascent peptides in the control of elongation for mycobacillin synthesis

“…We here report a continuation of our previous work on the functional characterization of constituent enzymes of the three-fraction enzyme mycobacillin synthetase, which showed that fraction A formed the first initiating complex with L-proline in the presence of ATP, that fraction B formed the second initiation complex with pentapeptide in the presence of ATP and that fraction C formed the third or last initiation complex with nonapeptide in the presence of ATP (Ghosh et al, 1985). The present work reports elongation studies demonstrating that fraction A converted tripeptide in the presence of ATP and the next amino acid in the sequence into tetrapeptide and, again, converted tetrapeptide into pentapeptide (also in the presence of ATP and the next amino acid in the sequence), thus lengthening the chain by one amino acid in the presence of ATP for the two successive steps.…”

“…The synthesis of mycobacillin (Majumder & Bose, 1958), a cyclotridecapeptide antibiotic (L-Pro-D-Asp-D- (Majumder & Bose, 1960), is catalysed by the three-fraction (A, B and C) enzyme complex mycobacillin synthetase (Ghosh et al, 1983), which was purified to homogeneity (Ghosh et al, 1986). The synthesis occurs by three-step process, the enzyme fraction A synthesizing pentapeptide in the first step, fraction B the nonapeptide in the second step, and fraction C completing the molecules in the third step (Ghosh et al, 1985). None of the fractions contains the pantothenic acid arm , but instead a sequential activation of an amino-aciddependent ATP,--[32P]PI exchange reaction (Sengupta & Bose, 1972, 1974 is carried out instead of individual activation, as is the case for other bioactive peptides (Itoh et al, 1968;Gevers et al, 1968).…”

Characterization of three-fraction mycobacillin synthetase

“…Finally each of the active fractions was concentrated and separately layered over a discontinuous sucrose gradient (10%, 12.5%, 15%, 17.5 %, 20o%, 22.5 %, w/v) and centrifuged for 8 h at 40000 rev./min in Beckman SW 40 rotor at 0 'C. All the subsequent studies were performed with these active fractions (Ghosh et al, 1985).…”