Proof of the Structure of the Stemodia chilensis Tetracyclic Diterpenoid (+)-19-Acetoxystemodan-12-ol by Synthesis from (+)-Podocarpic Acid: X-ray Structure Determination of a Key Intermediate.

The first synthesis of (+)-19-acetoxystemodan-12-ol (1), a stemodane diterpenoid isolated from Stemodia chilensis, is described. The structure was supported by an X-ray crystallographic analysis of intermediate (+)-9a, which confirmed the proposed structure and excluded the structure of (-)-19-hydroxystemod-12-ene as a possible candidate for the Chilean Calceolaria diterpenoid to which the (-)-19-hydroxystemar-13-ene structure (9b) had been erroneously assigned.

Studies on synthetic LuxR solo hybrids.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

Functional and structural study of the dimeric inner membrane protein SbmA.

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

Regio- and diastereoselective synthesis and X-ray structure determination of (+)-2-deoxyoryzalexin S from (+)-podocarpic acid. Structural nonidentity with its nominal natural isolated enantiomer.

(+)-2-Deoxyoryzalexin S (1), the nominal enantiomer of a diterpenoid isolated in Chile from Calceolaria species, was regio- and diastereoselectively synthesized from (+)-podocarpic acid. (+)-2-Deoxyoryzalexin S (1) was characterized also as its acetyl derivative, (+)-2, whose structure was confirmed by X-ray crystallographic analysis. Surprisingly, comparison of the data recorded for (+)-1 and (+)-2 and those reported in the literature for the Calceolaria isolated diterpenoid 1 and its derivative (-)-2 showed some differences, suggesting that the latter do not possess the proposed structures.