A fluorometric assay for high-throughput phosphite quantitation in biological and environmental matrices.

Phosphite, the anion of phosphorus acid, is an important metabolite in the global biogeochemical phosphorus cycle and a phosphorus species with unique agricultural properties. As such, methods for detecting phosphite quantitatively and selectively are critical to evidencing phosphorus redox chemistry. Here, we present a fluorescence-based assay for phosphite, utilizing the NAD+-dependent oxidation of phosphite by phosphite dehydrogenase and the subsequent reduction of resazurin to resorufin. With the application of a thermostable phosphite dehydrogenase, a medium-invariant analytical approach, and novel sample preparation methods, the assay is capable of rapid and accurate phosphite quantification with a 3 µM limit of detection in a wide array of biologically- and environmentally-relevant matrices, including bacterial and archaeal cell lysate, seawater, anaerobic digester sludge, and plant tissue. We demonstrate the utility of the assay by quantitating phosphite uptake in a model crop plant in the presence or absence of a phosphite-oxidising strain of Pseudomonas stutzeri as a soil additive, establishing this bacterium as an efficient phosphite converting biofertilizer.

Selenocysteine substitutions in thiyl radical enzymes.

Cysteine thiyl radicals are implicated as cofactors in a variety of enzymatic transformations, as well as transient byproducts of oxidative stress, yet their reactivity has undermined their detailed study. Selenocysteine exhibits a lower corresponding selenyl radical reduction potential, thus taming this radical reactivity without significant steric perturbation, potentially affording a glimpse into otherwise fleeting events in thiyl radical catalysis. In this chapter, we describe a suite of fusion protein constructs for general and efficient production of site-specifically incorporated selenoproteins by a recently developed nonsense suppression technology. As a proof of concept, we produced NikJ, a member of the radical S-adenosyl methionine enzyme family involved in the biosynthesis of peptidyl nucleoside antibiotics. We place emphasis throughout the plasmid assembly, protein expression, and selenium quantitation on accommodating the structural and functional diversity of thiyl radical enzymes. The protocol produces NikJ with near quantitative selenocysteine insertion, 50% nonsense read-through, and facile protein purification.