The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5‐diketopiperazines (2,5‐DKPs) have been widely developed ...by medicinal chemists, but their biological production is yet to be exploited. We introduce an in vivo method for incorporating non‐canonical amino acids (ncAAs) into 2,5‐DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5‐DKP biosynthetic pathways. CDPSs use aminoacyl‐tRNAs as substrates. We exploited the natural ability of aminoacyl‐tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non‐canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5‐DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide‐tailoring enzymes found in 2,5‐DKP biosynthetic pathways.
Nichtnatürlich und doch natürlich: Cyclodipeptid‐Synthasen (CDPSs) sind Enzyme, die Aminoacyl‐tRNAs aus der Translationsmaschinerie abzweigen, um Cyclodipeptide herzustellen. Die natürliche Promiskuität von Aminoacyl‐tRNA‐Synthetasen wurde genutzt, um zu zeigen, dass CDPSs ein breites Spektrum nichtkanonischer Aminosäuren als Substrate tolerieren. So gelang die Biosynthese von ca. 200 Cyclodipeptiden mit nichtkanonischen Aminosäuren.
Cyclodipeptide synthases (CDPSs) are small enzymes structurally related to class-I aminoacyl-tRNA synthetases (aaRSs). They divert aminoacylated tRNAs from their canonical role in ribosomal protein ...synthesis, for cyclodipeptide formation. All the CDPSs experimentally characterized to date are bacterial. We show here that a predicted CDPS from the sea anemone
Nematostella vectensis is an active CDPS catalyzing the formation of various cyclodipeptides, preferentially containing tryptophan. Our findings demonstrate that eukaryotes encode active CDPSs and suggest that all CDPSs have a similar aminoacyl-tRNA synthetase-like architecture and ping-pong mechanism. They also raise questions about the biological roles of the cyclodipeptides produced in bacteria and eukaryotes.
► New cyclodipeptide synthase (CDPS) with new substrate specificity ► CDPSs are found in animals ► Nonribosomal peptide synthesis in animals ► Bacterial and eukaryotic CDPSs, a same mechanism with an aminoacyl-enzyme intermediate
The core light‐harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n ‐octyl‐β‐ d ‐glucopyranoside into smaller subunit forms, exhibiting a dramatic ...blue‐shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n ‐octyl‐β‐D‐glucopyranoside, we have previously shown that the B820 form is not only constituted by the αβ heterodimer alone, but that it exists in an equilibrium between the αβ heterodimer and β 2 homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoritical model for αβ and β 2 , we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non‐covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.
The core light-harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n-octyl-beta-D-glucopyranoside into smaller subunit forms, exhibiting a dramatic ...blue-shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n-octyl-beta-D-glucopyranoside, we have previously shown that the B820 form is not only constituted by the alphabeta heterodimer alone, but that it exists in an equilibrium between the alphabeta heterodimer and beta(2) homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoretical model for alphabeta and beta(2), we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non-covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.
The core light-harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n -octyl-beta-d-glucopyranoside into smaller subunit forms, exhibiting a dramatic ...blue-shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n -octyl-beta-D-glucopyranoside, we have previously shown that the B820 form is not only constituted by the αbeta heterodimer alone, but that it exists in an equilibrium between the αbeta heterodimer and beta2 homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoritical model for αbeta and beta2, we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non-covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes. PUBLICATION ABSTRACT
The core light‐harvesting LH1 protein from
Rhodospirillum rubrum
can dissociate reversibly in the presence of
n
‐octyl‐β‐
d
‐glucopyranoside into smaller subunit forms, exhibiting a dramatic ...blue‐shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of
n
‐octyl‐β‐D‐glucopyranoside, we have previously shown that the B820 form is not only constituted by the αβ heterodimer alone, but that it exists in an equilibrium between the αβ heterodimer and β
2
homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoritical model for αβ and β
2
, we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non‐covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.
Light harvesting complexes 2 (LH2) are the peripheral antenna proteins in the bacterial photosynthetic apparatus and are built of α/β-heterodimers containing three bacteriochlorophylls and two ...carotenoids each. Previously, we have found in 2D-crystals that the complexes could be inserted within the membrane with a tilt with respect to the membrane plane (
Rhodobacter sphaeroides) or without tilt (
Rubrivivax gelatinosus). To investigate whether the tilted insertion represents the native state or if it is due to specific 2D-crystal contacts, we have used atomic force microscopy to investigate LH2 from
Rhodopseudomonas acidophila reconstituted at different lipid to protein ratios. High-resolution topographs could be acquired of two types of 2D-crystals or of densely packed membranes. Interestingly, in type 2 2D-crystals and in non-crystalline densely packed membranes, cylinders are integrated with their symmetry axis normal to the membrane plane, while in type 1 2D-crystals LH2 cylinders are integrated with a tilt of ∼4° with respect to the membrane plane. Therefore, we present strong evidence that the tilt of LH2 does not represent the native membrane state and is due to protein–protein contacts in specific 2D-crystals.
We have studied the equilibrium between the dissociated B777 form (absorbing at 777 nm) of the light-harvesting complex of
Rhodospirillum rubrum and the oligomeric B820 form. Analysis of the reaction ...order for the B820 dissociation reaction to form B777 shows that this reaction depends on the concentration of octylglucoside detergent (
n-octyl-β-
D-glucopyranoside (βOG)) present in the sample. At low βOG concentrations (less than 1.2%) this reaction requires two components, presumably one α-B777 and one β-B777, implying that the B820 subunit is a dimer. At higher βOG concentrations this reaction requires four components, implying that B820 is a tetramer. These results partly explain the discrepancies in the literature about the stoichiometry of B820 and open an original way for studying protein–detergent interactions.
The Hfq (Host factor 1) polypeptide is a nucleic acid binding protein involved in the synthesis of many polypeptides. Hfq particularly affects the translation and the stability of several RNAs. In an ...earlier study, the use of fold recognition methods allowed us to detect a relationship between Escherichia coli Hfq and the Sm topology. This topology was further validated by a series of biophysical studies and the Hfq structure was modelled on an Sm protein. Hfq forms a β‐sheet ring‐shaped hexamer. As our previous study predicted a large number of alternative conformations for the C‐terminal region, we have determined whether the last 19 C‐terminal residues are necessary for protein function. We find that the C‐terminal truncated protein is fully capable of binding a polyadenylated RNA (Kd of 120 pm vs. 50 pm for full‐length Hfq). This result shows that the functional core of E. coli Hfq resides in residues 1–70 and confirms previous genetic studies. Using equilibrium unfolding studies, however, we find that full‐length Hfq is 1.8 kcal·mol−1 more stable than its truncated variant. Electron microscopy analysis of both truncated and full‐length proteins indicates a structural rearrangement between the subunits upon truncation. This conformational change is coupled to a reduction in β‐strand content, as determined by Fourier transform infra‐red. On the basis of these results, we propose that the C‐terminal domain could protect the interface between the subunits and stabilize the hexameric Hfq structure. The origin of this C‐terminal domain is also discussed.
PDF
