R97 at "Handlebar" Binding Mode in Active Pocket Plays an Important Role in Fe(II)/α-Ketoglutaric Acid-Dependent Dioxygenase cis-P3H-Mediated Selective Synthesis of (2S,3R)-3-Hydroxypipecolic Acid.

Pipecolic acid (Pip) and its derivative hydroxypipecolic acids, such as (2S,3R)-3-hydroxypipecolic acid (cis-3-L-HyPip), are components of many natural and synthetic bioactive molecules. Fe(II)/α-ketoglutaric acid (Fe(II)/2-OG)-dependent dioxygenases can catalyze the hydroxylation of pipecolic acid. However, the available enzymes with desired activity and selectivity are limited. Herein, we compare the possible candidates in the Fe(II)/2-OG-dependent dioxygenase family, and cis-P3H is selected for potentially catalyzing selective hydroxylation of L-Pip. cis-P3H was further engineered to increase its catalytic efficiency toward L-Pip. By analyzing the structural confirmation and residue composition in substrate-binding pocket, a "handlebar" mode of molecular interactions is proposed. Using molecular docking, virtual mutation analysis, and dynamic simulations, R97, E112, L57, and G282 were identified as the key residues for subsequent site-directed saturation mutagenesis of cis-P3H. Consequently, the variant R97M showed an increased catalytic efficiency toward L-Pip. In this study, the kcat/Km value of the positive mutant R97M was about 1.83-fold that of the wild type. The mutation R97M would break the salt bridge between R97 and L-Pip and weaken the positive-positive interaction between R97 and R95. Therefore, the force on the amino and carboxyl groups of L-Pip was lightly balanced, allowing the molecule to be stabilized in the active pocket. These results provide a potential way of improving cis-P3H catalytic activity through rational protein engineering.

Gold nanoparticle based photometric determination of tobramycin by using new specific DNA aptamers.

A magnetic bead-based SELEX was applied to identify 37 single-stranded DNA aptamers specific for tobramycin after ten rounds of selection. The aptamers were classified into nine families according to sequence analysis. Among them, several aptamers with typical sequences were selected and their dissociation constants (Kds) were determined by a fluorescent method. An aptamer termed "Ap 32", with a Kd value of 56.8 ± 4.6 nM, possesses the highest affinity and satisfactory specificity. Theoretical modeling showed that nucleotides 14-18 and 26-29 play a most significant role in the interaction between aptamer and tobramycin. Subsequently, the sequence of Ap 32 was optimized through rationally designed truncation. The truncated aptamer Ap 32-2 consists of 34 nucleotides and has a Kd that is similar to the original one. It was chosen as the optimal aptamer for use in the assay and was immobilized on gold nanoparticles. On addition of tobramycin, the color turns from red to purple. The findings were used to design a photometric assay (best performed at 520 nm) that has a linear response in the 100 nM to 1.4 µM concentration range, with a 37.9 nM detection limit. The method was successfully applied to the determination of tobramycin in (spiked) honey samples. Graphical abstract A 34-nucleotide aptamer specific for tobramycin was obtained through magnetic beads-based systematic evolution of ligands by exponential enrichment (SELEX) and structural analysis-based rational post-SELEX truncation, and then applied to the determination of tobramycin using a gold nanoparticle-based photometric assay.