ABSTRACT
Two new classes of inhibitors of LpPLA2 have been identified in fermentations of Pseudomonas fluorescens. The two structurally isomeric series differ in the geometry of closure of the bicyclic carbamate and comprise a range of compounds varying only in length of their lipophilic sidechain. The most abundant species were extracted from the cells and purified by silica and C18 chromatography. Members of the more stable class were shown to be potent and selective competitive inhibitors of LpPLA2.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/isolation & purification , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Phospholipases A/antagonists & inhibitors , Pseudomonas fluorescens/metabolism , Pyrans/isolation & purification , Pyrans/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Animals , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/metabolism , Fermentation , Humans , Phospholipases A2 , Pyrans/metabolism , RabbitsABSTRACT
Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.
Subject(s)
4-Butyrolactone/isolation & purification , Anti-Bacterial Agents/biosynthesis , Streptomyces/chemistry , 4-Butyrolactone/chemical synthesis , 4-Butyrolactone/chemistry , Chromatography, High Pressure Liquid , Stereoisomerism , Streptomyces/drug effects , Streptomyces/metabolismABSTRACT
In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.
Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/genetics , Isoleucine/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Transfer, Leu/genetics , Base Sequence , Codon , Escherichia coli/metabolism , Molecular Sequence Data , RNA, Transfer, Leu/metabolism , Recombinant Proteins/biosynthesisABSTRACT
Human interleukin-4 (IL-4) is a member of the family of haemopoietic cytokines that modulate cell proliferation and differentiation within the immune system. It has a four-helix-bundle structure, and possesses a high degree of mobility in certain regions, notably in the two long loops running the length of the bundle in its up-up-down-down topology. Information from a variety of three-dimensional heteronuclear NMR experiments, including chemical shifts, coupling constants and NOE data, is analysed in terms of the solution structure of IL-4. In addition, structure calculations with and without specific restraints such as hydrogen bond location or torsion angle restrictions are compared in the light of the dynamic behaviour of the polypeptide chain. Particular emphasis is placed on defining the lengths and positions of secondary structure elements, and on the likely structural preferences within the less well ordered loop regions. The overall topology of IL-4 is compared with those defined in recent structure determinations of related proteins. This analysis is combined with recent mutagenesis data to propose a possible mode of interaction of IL-4 with its receptor.
Subject(s)
Interleukin-4/chemistry , Crystallography, X-Ray , Humans , Hydrogen Bonding , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Protein Structure, SecondaryABSTRACT
Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity.
