Phosphate Monoester Hydrolysis in Cyclohexane

The hydrolysis of simple phosphate monoesters is among the most difficult reactions that are subject to catalysis by enzymes, and it has been suggested that extraction of the substrates from solvent water may contribute to the catalytic effects of phosphohydrolases. Here, we show that the tetrabutylammonium salt of neopentyl phosphate enters wet cyclohexane at concentrations sufficient to allow determination of its rate of hydrolysis. The second-order rate constant for hydrolysis of the phosphomonoester dianion is enhanced approximately 2 x 10(12)-fold by transfer from water to cyclohexane. That rate enhancement arises from an increase in the entropy of activation

The hydrolysis of phosphate diesters in cyclohexane and acetone

The hydrolysis of phosphate diesters is one of the most difficult reactions known. Here we show that in acetone or cyclohexane, at 25 °C, phosphodiesters undergo hydrolysis 5 × 105 and 2 × 109-fold more rapidly than in water, respectively, and that this rate enhancement is achieved by lowering the enthalpy of activation

The rate of spontaneous cleavage of the glycosidic bond of adenosine

Previous estimates of the rate of spontaneous cleavage of the glycosidic bond of adenosine were determined by extrapolating the rates of the acid - and base-catalyzed reactions to neutral pH. Here we show that cleavage also proceeds through a pH-independent mechanism. Rate constants were determined as a function of temperature at pH 7 and a linear Arrhenius plot was constructed. Uncatalyzed cleavage occurs with a rate constant of 3.7 × 10−12 s−1 at 25 °C, and the rate enhancement generated by the corresponding glycoside hydrolase is ~5 × 1012-fold

Dimerization mechanism of an inverted-topology ion channel in membranes

Many ion channels are multisubunit complexes where oligomerization is an obligatory requirement for function as the binding axis forms the charged permeation pathway. However, the mechanisms of in-membrane assembly of thermodynamically stable channels are largely unknown. Here, we demonstrate a key advance by reporting the dimerization equilibrium reaction of an inverted-topology, homodimeric fluoride channel Fluc in lipid bilayers. While the wild-type channel is a long-lived dimer, we leverage a known mutation, N43S, that weakens N

Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonasmendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today

Still rocking in the structural era: A molecular overview of the small multidrug resistance (SMR) transporter family

The small multidrug resistance (SMR) family is composed of widespread microbial membrane proteins that fulfill different transport functions. Four functional SMR subtypes have been identified, which variously transport the small, charged metabolite guanidinium, bulky hydrophobic drugs and antiseptics, polyamines, and glycolipids across the membrane bilayer. The transporters possess a minimalist architecture, with ∼100-residue subunits that require assembly into homodimers or heterodimers for transport. In part because of their simple construction, the SMRs are a tractable system for biochemical and biophysical analysis. Studies of SMR transporters over the last 25 years have yielded deep insights for diverse fields, including membrane protein topology and evolution, mechanisms of membrane transport, and bacterial multidrug resistance. Here, we review recent advances in understanding the structures and functions of SMR transporters. New molecular structures of SMRs representing two of the four functional subtypes reveal the conserved structural features that have permitted the emergence of disparate substrate transport functions in the SMR family and illuminate structural similarities with a distantly related membrane transporter family, SLC35/DMT