Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis
10.1128/jb.177.18.5199-5205.1995
Crossref journal-article
American Society for Microbiology
Journal of Bacteriology (235)
Abstract

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.

Bibliography

Thibaut, D., Ratet, N., Bisch, D., Faucher, D., Debussche, L., & Blanche, F. (1995). Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis. Journal of Bacteriology, 177(18), 5199–5205.

Authors 6
  1. D Thibaut (first)
  2. N Ratet (additional)
  3. D Bisch (additional)
  4. D Faucher (additional)
  5. L Debussche (additional)
  6. F Blanche (additional)
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Dates
Type When
Created 8 years, 9 months ago (Nov. 9, 2016, 7:54 a.m.)
Deposited 4 years ago (July 29, 2021, 1:51 p.m.)
Indexed 11 months, 3 weeks ago (Sept. 3, 2024, 6:20 a.m.)
Issued 29 years, 11 months ago (Sept. 1, 1995)
Published 29 years, 11 months ago (Sept. 1, 1995)
Published Print 29 years, 11 months ago (Sept. 1, 1995)
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@article{Thibaut_1995, title={Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis}, volume={177}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.177.18.5199-5205.1995}, DOI={10.1128/jb.177.18.5199-5205.1995}, number={18}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Thibaut, D and Ratet, N and Bisch, D and Faucher, D and Debussche, L and Blanche, F}, year={1995}, month=sep, pages={5199–5205} }