Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes.

Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).

Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 A), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 A apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.

Synthesis and biological characterisation of [3H]BBL454, a new CCK2 selective radiolabelled agonist displaying original pharmacological properties.

[(3)H]BBL454, a new CCK(2) selective tritiated agonist was prepared via the reductive tritiation of a 5-aminopentyn-1-yl moiety introduced on the N-terminal end of a pentapeptide derivative of cholecystokinin. The binding properties of this labelled compound were determined on CHO cells transfected with the rat CCK(2) receptor. [(3)H]BBL454 is able to discriminate two affinity states of the CCK(2) receptor a supplementary indication of its validity for further exploring the heterogeneity of this receptor.

Picosecond dynamics of a peptide from the acetylcholine receptor interacting with a neurotoxin probed by tailored tryptophan fluorescence.

A tryptophan analog, dehydro-N-acetyl-L-tryptophanamide (delta-NATA), which is produced enzymatically via L-tryptophan 2',3'-oxidase from Chromobacterium violaceum, is newly used for time-resolved fluorescence. The absorption and emission maxima of delta-NATA at 332 and 417 nm, respectively, in 20% dimethylformamide-water are significantly shifted to the red with respect to those of tryptophan in water, permitting us to measure its fluorescence in the presence of tryptophan residues. We demonstrate that the steady-state spectra and the fluorescence decay of delta-NATA are very sensitive to environment, changing dramatically with solvent as the chromophore is localized within a protein and when this tagged protein binds to a peptide. The tryptophan oxidase was also used to modify the single Trp of a neurotoxin from snake (Naja nigricollis) venom. Modification of the toxin alpha (dehydrotryptophan-toxin alpha) permitted its investigation in complex with a synthetic 15-amino acid peptide corresponding to a loop of the agonist-binding site of acetylcholine receptor (AchR) from Torpedo marmorata species. The peptide alpha-185 possesses a single Trp at the third position (Trp187 of AchR) and a disulfide bridge between Cys192 and Cys193. A single-exponential rotational diffusion time with a constant of 1.65 ns is measured for the isolated 15-amino acid peptide. This suggests that Trp motion in the peptide in solution is strongly correlated with the residues downstream the peptide sequence, which may in part be attributed to long-range order imposed by the disulfide bond. The dynamics of the bound peptide are very different: the presence of two correlation times indicates that the Trp187 of the peptide has a fast motion (taur1 = 140 ps and r(0)1 = 0.14) relative to the overall rotation of the complex (taur2 = 3.4 ns and r(0)2 = 0.04). The correlation of the Trp residue with its neighboring amino acid residues and with the overall motion of the peptide is lost, giving rise to its rapid restricted motion. Thus, the internal dynamics of interacting peptides change on binding.

The albonoursin gene Cluster of S noursei biosynthesis of diketopiperazine metabolites independent of nonribosomal peptide synthetases.

Albonoursin [cyclo(deltaPhe-DeltaLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of alpha,beta unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP derivatives.