Bacitracin synthetase

Fr/ ysnov, The production of bacitracin synthetase bv Bacillus licheniformis ATCC 10716, FEBS... 

Bacitracin synthetase consists of three multifunctional enzymes A, B and C (Ref. 22, 32) which occur simultaneously for a short period in the growth phase (Ref. 35). Later in growth bacitracin synthetase becomes predominately cell bound. This may represent a natural immobilization of an enzyme complex resulting in a protection against proteolysis. Ultrogel ACA 34 gel filtration indicates that the molecular weights are 300 000, l80 000 and 330 000 Daltons respectively (unpublished results).(See Pig. 5)... 

All the enzymes of bacitracin synthetase contain the cofactor phosphopantetheine which probably acts as a covalently linked carrier of intermediates in the biosynthesis of bacitracin (Ref. 36, 37). 

Figure 5 Molecular mass estimation of peptide synthetases in one 3% SDS-PAGE. The molecular masses of the reference proteins recombinant tyrocidine synthetase 2 (190 kDa). myosin (205 kDa), and ACV synthetase (420 kD) are indicated. Extrapolation results in molecular masses of 570 kDa for gramicidin synthetase 2 (lane 1), 310,730, and 870 kDa for bacitracin synthetases 2, 1, and 3 (lane 2), 640 and 880 kDa for linear gramicidin synthetase 2 and tyrocidine synthetase 3 (lane 3), 1530 kDa for cyclosfwrin synthetase (lane 4), and 1460 kDa for peptolide SDZ 214-103 synthetase (lane 5). (From Ref. 58, with permission.)...

Bacitracin biosynthesis requires a non-ribosomal peptide synthetase with three major protein components, BacABC. This synthetase has a modular structure. There are associated regulatory and transport systems. Biosynthesis of bacitracin has been engineered in the surrogate host, B. subtilis, by genetic techniques. A strain, B. subtilis KE 350, expresses the entire 49-kb bacitracin... 

The poly (ribitol phosphate) synthetase and poly (glycerol phosphate) synthetase are inhibited by vancomycin, novobiocin, and Crystal Violet. Other antibiotic substances which interfere with cell-wall synthesis (such as bacitracin, ristocetin, and streptomycin) are almost without effect on the isolated synthetases, and penicillin is inhibitory at high concentrations only. Moreover, penicillin, vancomycin, and bacitracin do not markedly inhibit synthesis of cell-wall glycosaminopeptide in vitro, although the synthetical activity of extracts of cells which have been pretreated with these antibiotics is lowered.Convincing evidence that the primary site of inhibition by antibiotics is the biosynthesis of cell-wall material has been obtained only for the penicillins and cycloserine, and it appears that the action of even those antibiotics may be more complex than was originally supposed. 

Figure 3 The nonribosomal machinery (a) needed to produce bacitracin (b) consists of three separate synthetases BacA, BacB, and BacC, composing a total of 12 modules and 40 catalytic domains. These synthetases specifically interact with each other to produce the nonribosomal peptide bacitracin.

The amino acids in bacitracin biosynthesis are activated as aminoacyl adenylates before being transferred to separate thiol sites on the synthetase (Ref. 31, 34). See Pig 4. 

During bacitracin synthesis, intermediate peptides are thioester linked to the synthetase (Ref. 22). The peptides were... 

These heterocycles can occur as such in the product, as in the epothilones leinamycin and bacitracin, and as a tandem dimer as in the case of BLM, pyoche-lin, and yersiniabactin. These metabolites, biosynthesized by NRPS-PKS hybrid synthetases, are composed of a polyketide backbone featuring incorporated amino acids in the case of a PKS/NRPS hybrid or a peptidyl chain harboring ketone groups characteristic of an NRPS-PKS hybrid. 

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