Subject(s)
Anti-Bacterial Agents/biosynthesis , Bacteria/metabolism , Amino Acids/chemistry , Amino Acids/metabolism , Aminoglycosides , Bacteria/genetics , Catalysis , Macrolides , Molecular Conformation , Molecular Structure , Multienzyme Complexes/metabolism , Multigene Family , Mycobacteriaceae/genetics , Mycobacteriaceae/metabolism , Myxococcales/genetics , Myxococcales/metabolism , Pseudomonas/genetics , Pseudomonas/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Structure-Activity RelationshipABSTRACT
Measurement of nuclear Overhauser enhancement is one of the mainstays of structural studies of molecules in solution. Measurements, particularly over long distances, are often compromised by spin diffusion. A robust and accurate method for measuring nuclear Overhauser enhancements that are largely free of spin diffusion effects is presented. The effects of using imperfect radiofrequency pulses are considered and experimental data are presented for Amphotericin B.
Subject(s)
Magnetic Resonance SpectroscopySubject(s)
Anti-Bacterial Agents/biosynthesis , Ethers/metabolism , Macrolides/metabolism , Multienzyme Complexes/metabolism , Polyenes/metabolism , Animals , Ethers/chemistry , Fungi/metabolism , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Macrolides/chemistry , Plants/metabolism , Polyenes/chemistryABSTRACT
The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Mixed Function Oxygenases/genetics , Nocardiaceae/enzymology , Thiolester Hydrolases/genetics , Amino Acid Sequence , Bacterial Proteins , Base Sequence , Chymotrypsin , Cloning, Molecular , Cytochrome P-450 Enzyme System/isolation & purification , Escherichia coli/enzymology , Mixed Function Oxygenases/isolation & purification , Molecular Sequence Data , Mutagenesis, Site-Directed , Nocardiaceae/genetics , Oligonucleotide Probes , Open Reading Frames , Peptide Fragments/isolation & purification , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Restriction Mapping , Thiolester Hydrolases/biosynthesis , Thiolester Hydrolases/isolation & purification , TrypsinABSTRACT
Ethyl (Z)-16-phenylhexadec-9-enoate (3), an analog of ethyl oleate (2), was synthesized and added to cultures of Streptomyces cellulosae ATCC 12625 which normally produce fungichromin (1) as the principal polyene antibiotic. These cultures showed drastic reduction of fungichromin biosynthesis but afforded four new polyene antibiotics with a truncated four carbon side chain which are designated as isochainin (11) (an isomer of chainin (10], 14-hydroxyisochainin (12), 1'-hydroxyisochainin (13), and 1',14-dihydroxyisochainin (14). The close correspondence of 13C NMR chemical shifts between these compounds and fungichromin suggests that the stereochemistry at every site is exactly analogous.
