Roger Genet

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 Å), 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 Å 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.

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.

Captopril inhibits in vitro and in vivo the proliferation of primitive haematopoietic cells induced into cell cycle by cytotoxic drug administration or irradiation but has no effect on myeloid leukaemia cell proliferation

Angiotensin I‐converting enzyme (ACE) has been shown to be involved in the catabolism of the tetrapeptide acetyl‐Ser–Asp–Lys–Pro (AcSDKP). As AcSDKP is a physiological inhibitor of haematopoietic stem cell proliferation, we investigated the in vitro and in vivo effects of captopril, one of the specific inhibitors of ACE, on the proliferation of primitive haematopoietic cells. Regenerating bone marrow cells obtained from mice given one injection of cytosine arabinoside (100 mg/kg) as well as SA2 myeloid leukaemia cells were incubated in vitro for 24 h with 10−6 m captopril. Captopril significantly reduced the proportion of high proliferative potential colony‐forming cells (HPP‐CFC‐1) in S‐phase, whereas it had no effect on the proportion of SA2 leukaemic colony‐forming cells in S‐phase. When given in vivo to mice 1 h after 2 Gy γ‐irradiation or cytosine arabinoside (AraC) injection, captopril (100 mg/kg) was shown to prevent HPP‐CFC‐1 entry into S‐phase induced by these cytotoxic treatments. The observed effects correlated with a reduction in ACE degradative activity and an increase in the level of endogenous AcSDKP both in the supernatants of captopril‐treated bone marrow cells and in plasma of treated animals. The present findings suggest that AcSDKP might mediate the observed in vitro and in vivo inhibitory effects of captopril on primitive haematopoietic cell proliferation.

Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

EZ-configurational assignment of N-acetyl-α,β-dehydrotryptophan ethyl ester produced by L-tryptophan 2′,3′-oxidase from Chromobacterium violaceum

Abstract The steric configuration of the α,β-dehydrotryptophanyl moiety produced by incubation of N-acetyl-L-tryptophan ethyl ester with L-tryptophan 2′,3′-oxidase was investigated by 1H-NMR spectroscopy. It adopts unambiguously a Z-geometry.

Picosecond Dynamics of a Peptide from the Acetylcholine Receptor Interacting with a Neurotoxin Probed by Tailored Tryptophan Fluorescence

Abstract A tryptophan analog, dehydro-N-acetyl-l-tryptophanamide (Δ-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 Δ-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 Δ-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 α (dehydrotryptophan-toxin α) 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 α-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 (τr1 = 140 ps and r(0)1 = 0.14) relative to the overall rotation of the complex (τr2 = 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.

Asymmetric deuteration of N-acetyl-(Z)-α,β-dehydrotryptophan-(L)-phenylalanine methyl ester produced by (L)-tryptophan 2′,3′-oxidase from Chromobacterium violaceum. A new route for stereospecific labelling of peptides

Abstract A novel approach to the synthesis of labelled ( 2 H, 3 H) peptides through the catalytic asymmetric reduction of Δ Z Trp containing peptides, using rhodium complexes with chiral diphosphine ligands as the catalysts, is described. Ac-Δ Z Trp-( L )-Phe-OMe is used as a model substrate to study this new route. The (Z)-α,β-dehydropeptide is produced by ( L )-tryptophan 2′,3′-oxidase from Chromobacterium violaceum in a single step reaction. Diastereomeric excesses up to 98 % have been obtained with ( R , R )-dipamp as ligand in the catalyst. Extremely high stereoselectivities for producing the ( L , L )- or ( D , L )-isomer could be achieved using the appropriate chiral ligands. This method has good potential for stereospecific labelling (deuteration or tritiation) of peptides.

Diastereoselective deuteration of (Z)-α,β-dehydrotryptophanyl-containing biological peptides controlled by chiral rhodium catalysts

Abstract The regio- and stereoselective labelling ( 2 H, 3 H) of tryptophanyl containing biological peptides catalysed by chiral rhodium complexes is described. The Δ Z Trp-residue is directly produced in the peptide by a catalytic process, involving the ( S )-tryptophan 2′,3′-oxydase from Chromobacterium violaceum . Two biological dehydropentapeptides, pGlu-His-Δ Z Trp-Ser-Tyr and Boc-βAla-Δ Z Trp-Met-Asp-Phe-NH 2 , have been thus synthesized. Asymmetric deuteration of these compounds was then investigated in the presence of various rhodium catalysts. High stereocontrol was achieved by suitable choice of the chiral ligands. This strategy offers new possibilities for the asymmetric synthesis of labelled ( 2 H, 3 H) peptides without ultimate deprotection step.

Asymmetric Reduction of (Z)-α,β-Dehydrotryptophanyl-containing Biological Peptides with Sulfur Functionality. Influence of Substrate on Stereoselectivity

Abstract Two (Z)-α,β-dehydropentapeptides, Boc-βAla-ΔZ Trp-Met-Asp-Phe-NH2 1 and Boc-βAlaΔZ-Trp-NLeu-Asp-Phe-NH2 2, have been synthesized by enzymatic α,β-dehydrogenation, and then submitted to rhodium-catalysed asymmetric hydrogenation. The influence of the S-atom on the rate and stereoselectivity of the reduction have been investigated.