Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.

Natural nonproteinogenic amino acids vastly outnumber the well-known 22 proteinogenic amino acids. Such amino acids are generated in specialized metabolic pathways. In these pathways, diverse biosynthetic transformations, ranging from isomerizations to the stereospecific functionalization of C–H bonds, are employed to generate structural diversity. The resulting nonproteinogenic amino acids can be integrated into more complex natural products. Here we review recently discovered biosynthetic routes to freestanding nonproteinogenic α-amino acids, with an emphasis on work reported between 2013 and mid-2019.

Nitroimidazoles are one of the most effective ways to treat anaerobic bacterial infections.S ynthetic nitroimidazoles are inspired by the structure of azomycin, isolated from Streptomyces eurocidicus in 1953. Despite its foundational role,nobiosynthetic gene cluster for azomycin has been found. Guided by bioinformatics,wei dentified ac ryptic biosynthetic gene cluster in Streptomyces cattleya and then carried out in vitro reconstitution to deduce the enzymatic steps in the pathway linking l-arginine to azomycin. The gene cluster we discovered is widely distributed among soil-dwelling actinobacteria and proteobacteria, suggesting that azomycin and related nitroimidazoles may play important ecological roles. Our work sets the stage for development of biocatalytic approaches to generate azomycin and related nitroimidazoles.

Copper indium disulphide (CIS) nanocrystals (NCs) were prepared using a one-pot synthesis. The stoichiometry was optimized based on its current density as measured by photoelectrochemical (PEC) experiments at interfaces between NC films deposited on ITO and 0.1 M methyl viologen dichloride (MV(2+)) solution. This method also offers insight into the kinetics of the photoreaction. A copper poor sulphur rich starting ratio was found to produce a copper-rich, indium-poor and slightly sulphur rich material. Further NC characterization was performed with SEM and TEM to investigate the morphology and crystallinity of the 30-70 nm NCs. The oxidation states of the individual elements were determined to be I, III, and 2- for Cu, In and S, respectively. Characteristics of optimal as-prepared NCs were found to be compatible among high functioning absorbing layers.

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to ( S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of HO. To understand this unusual chemistry, we obtain ∼1.5 Å resolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the ( S)-configured product. Together with our earlier studies on an O, PLP-dependent l-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from l-arginine.

Nitroimidazoles are one of the most effective ways to treat anaerobic bacterial infections. Synthetic nitroimidazoles are inspired by the structure of azomycin, isolated from Streptomyces eurocidicus in 1953. Despite its foundational role, no biosynthetic gene cluster for azomycin has been found. Guided by bioinformatics, we identified a cryptic biosynthetic gene cluster in Streptomyces cattleya and then carried out in vitro reconstitution to deduce the enzymatic steps in the pathway linking l‐arginine to azomycin. The gene cluster we discovered is widely distributed among soil‐dwelling actinobacteria and proteobacteria, suggesting that azomycin and related nitroimidazoles may play important ecological roles. Our work sets the stage for development of biocatalytic approaches to generate azomycin and related nitroimidazoles.

No abstract

The organic cofactor pyridoxal phosphate (PLP) is used by enzymes to catalyze numerous modifications of amino acid substrates. Recently, the diversity of PLP‐dependent enzymes has been expanded with the discovery of several enzymes that use O2 as a co‐substrate to perform chemically challenging oxidative reactions. The interesting chemistry catalyzed by such O2‐, PLP‐dependent oxidases prompted us to undertake a bioinformatic search for new O2‐, PLP‐dependent enzymes. We identified one widely distributed enzyme, which we named RohP. Through in vitro biochemical studies we characterized the activity of RohP and found that it uses PLP and O2 to catalyze the hydroxylation of an sp3‐hybridized carbon in l‐arginine, giving 4‐hydroxy‐2‐ketoarginine, along with stoichiometric conversion of O2 to H2O2. Surprisingly, we found that the hydroxyl group in the 4‐hydroxy‐2‐ketoarginine product is derived from water, raising questions about the mechanism of RohP. To provide insight into the mechanism behind this hydroxylation, we used X‐ray crystallography to obtain four ~1.5 Å resolution snapshots interpreted to represent RohP at different stages of its catalytic cycle: the holo enzyme, two different PLP‐bound intermediates, and the enzyme in complex with (S)‐4‐hydroxy‐2‐ketoarginine. These structures reveal that the dynamic N‐terminus of RohP, which is required for catalysis, becomes ordered upon substrate binding, sealing off the active site from the bulk solvent. Concurrently, several conformational changes of residues in the active site help to properly orient the substrate for catalysis. Through our structural work we also identify a conserved histidine, which we demonstrate is essential for the later steps in catalysis, and we suggest that this residue may be key to catalyzing a stereospecific alkene hydration. Collectively, our biochemical and X‐ray crystallographic investigation provides insights into how O2‐, PLP‐dependent oxidases can catalyze the challenging hydroxylation of an unactivated sp3‐hybridized carbon in l‐arginine and sets the stage for further mechanistic studies on this exciting group of enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.