Structural Basis of Substrate Promiscuity and Catalysis by the Reverse Prenyltransferase -Dimethylallyl-l-tryptophan Synthase from .

The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C-C, C-N, and C-O bond-forming trajectories. DMATS1 from catalyzes the reverse N-prenylation of l-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of l-Trp.

Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable a sensitive NMR technique known as hyperpolarized Xe chemical exchange saturation transfer (hyper-CEST).

Detecting protein-protein interactions by Xe-129 NMR.

Detection of protein-protein interactions (PPIs) is limited by current bioanalytical methods. A protein complementation assay (PCA), split TEM-1 β-lactamase, interacts with xenon at the interface of the TEM-1 fragments. Reconstitution of TEM-1-promoted here by cFos/cJun leucine zipper interaction-gives rise to sensitive 129Xe NMR signal in bacterial cells.

Structure and Function of the Acetylpolyamine Amidohydrolase from the Deep Earth Halophile .

Polyamines are small organic cations that are essential for cellular function in all kingdoms of life. Polyamine metabolism is regulated by enzyme-catalyzed acetylation-deacetylation cycles in a fashion similar to the epigenetic regulation of histone function in eukaryotes. Bacterial polyamine deacetylases are particularly intriguing, because these enzymes share the fold and function of eukaryotic histone deacetylases.

Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by CymD.

Indole prenyltransferases catalyze the prenylation of l-tryptophan (l-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from catalyzes the reverse N1 prenylation of l-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation.

A Structural Basis for Xe Hyper-CEST Signal in TEM-1 β-Lactamase.

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 β-lactamase (bla) and maltose binding protein (MBP).

Cryptophane Nanoscale Assemblies Expand Xe NMR Biosensing.

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution.

Xenon-Protein Interactions: Characterization by X-Ray Crystallography and Hyper-CEST NMR.

The physiological activity of xenon has long been recognized, though the exact nature of its interactions with biomolecules remains poorly understood. Xe is an inert noble gas, but can act as a general anesthetic, most likely by binding internal hydrophobic cavities within proteins. Understanding Xe-protein interactions, therefore, can provide crucial insight regarding the mechanism of Xe anesthesia and potentially other general anesthetic agents.

Thermophilic Ferritin 24mer Assembly and Nanoparticle Encapsulation Modulated by Interdimer Electrostatic Repulsion.

Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at a low ionic strength, and the protein forms a hollow assembly of 24 protomers at a high ionic strength (∼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation.

A Genetically Encoded β-Lactamase Reporter for Ultrasensitive (129) Xe NMR in Mammalian Cells.

Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub-micromolar concentrations. Particularly useful are genetically encoded, single-protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Herein, we report TEM-1 β-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) (129) Xe NMR, with significant saturation contrast at 0.

Programming A Molecular Relay for Ultrasensitive Biodetection through (129)Xe NMR.

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized (129) Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]-Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding.