Single Stabilizing Point Mutation Enables High-Resolution Co-Crystal Structures of the Adenosine A Receptor with Preladenant Conjugates.

The G protein-coupled adenosine A receptor (A AR) is an important new (potential) drug target in immuno-oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A AR antagonists displaying exceptional selectivity. While crystal structures of the human A AR have been solved, mostly using the A -StaR2 protein that bears 9 point mutations, co-crystallization with Preladenant derivatives has so far been elusive.

Pristinol, a Sesquiterpene Alcohol with an Unusual Skeleton from Streptomyces pristinaespiralis.

A terpene cyclase from Streptomyces pristinaespiralis was characterized as the synthase for (+)-(2S,3S,9R)-pristinol. The structure of this sesquiterpene alcohol, which has a new carbon skeleton, was established by NMR spectroscopy and single-wavelength anomalous-dispersion X-ray crystallography. Extensive isotopic labelling experiments were performed to distinguish between various possible cyclization mechanisms of the terpene cyclase and to decipher the EI-MS fragmentation mechanism for pristinol.

Position-Specific Mass Shift Analysis: A Systematic Method for Investigating the EI-MS Fragmentation Mechanism of epi-Isozizaene.

The EI-MS fragmentation mechanism of the bacterial sesquiterpene epi-isozizaene was investigated through enzymatic conversion of all 15 synthetic ((13) C1 )FPP isotopomers with the epi-isozizaene synthase from Streptomyces albus and GC-MS and GC-QTOF analysis including MS-MS. A systematic method, which we wish to call position-specific mass shift analysis, for the identification of the full set of fragmentation reactions was developed.

A method for investigating the stereochemical course of terpene cyclisations.

Three sesquiterpene cyclases from Streptomyces scabei 87.22, Streptomyces venezuelae ATCC 10712 and Streptomyces clavuligerus ATCC 27064 were characterised and their products were identified as (-)-neomeranol B, (+)-isodauc-8-en-11-ol and (+)-intermedeol, respectively. The stereochemical courses of the terpene cyclisations were investigated by use of various (13)C-labelled FPP isotopomers.

Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted.

Synthesis and bioactivity of analogues of the marine antibiotic tropodithietic acid.

Tropodithietic acid (TDA) is a structurally unique sulfur-containing antibiotic from the Roseobacter clade bacterium Phaeobacter inhibens DSM 17395 and a few other related species. We have synthesised several structural analogues of TDA and used them in bioactivity tests against Staphylococcus aureus and Vibrio anguillarum for a structure-activity relationship (SAR) study, revealing that the sulfur-free analogue of TDA, tropone-2-carboxylic acid, has an antibiotic activity that is even stronger than the bioactivity of the natural product. The synthesis of this compound and of several analogues is presented and the bioactivity of the synthetic compounds is discussed.

Induced-fit mechanism in class I terpene cyclases.

We present crystallographic and functional data of selina-4(15),7(11)-diene synthase (SdS) from Streptomyces pristinaespiralis in its open and closed (ligand-bound) conformation. We could identify an induced-fit mechanism by elucidating a rearrangement of the G1/2 helix-break motif upon substrate binding. This rearrangement highlights a novel effector triad comprising the pyrophosphate sensor Arg178, the linker Asp181, and the effector Gly182-O.

Marine bacteria from the Roseobacter clade produce sulfur volatiles via amino acid and dimethylsulfoniopropionate catabolism.

Dimethylsulfoniopropionate (DMSP) is a versatile sulfur source for the production of sulfur-containing secondary metabolites by marine bacteria from the Roseobacter clade. (34)S-labelled DMSP and cysteine, and several DMSP derivatives with modified S-alkyl groups were synthesised and used in feeding experiments that gave insights into the biosynthesis of sulfur volatiles from these bacteria.

An improved technique for the rapid chemical characterisation of bacterial terpene cyclases.

A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2μ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli.