Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2‐indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14–76 nm ) and cellular potencies in the low nanomolar range (165–334 nm ) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co‐crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2–2.4 Å resolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non‐spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor‐bound ligands is discussed.     

Identification of a Hotspot Residue for Improving the Thermostability of a Flavin-Dependent Monooxygenase

HadA is a flavin-dependent monooxygenase that can catalyze the denitration and dehalogenation of a wide variety of toxicants such as pesticides. Although these enzymatic reactions are useful for bioremediation or biocatalysis, the application of HadA for these purposes is not yet possible because of its low thermostability. In this work we have engineered HadA to be more thermostable through the use of structural, in silico, and rational approaches. The X-ray structure of HadA was solved to obtain a reliable three-dimensional protein model for further prediction of thermostable variants. In silico analysis by using two bioinformatic tools—FireProt and Disulfide by Design—suggested 102 variants that we then further refined by applying rational criteria including the location of a particular residue and its nearby interactions, as well as other biophysical parameters to narrow down the list to six candidates. The G513Y variant was found to be an optimal engineered candidate because it has significantly improved stability relative to the wild-type enzyme and equivalent activity. G513Y has an activity half-life 72 (50 °C) and 160 times (45 °C) longer than that of the wild-type enzyme. Coupled together with thermostable reactions of reduced flavin and NADH-regenerating systems, the G513Y variant can be used to catalyze denitration of 4nitrophenol at 45 °C. Structure/sequence alignments of HadA and its homologues indicate that several flavin-dependent monooxygenases also contain amino acid residues homologous to the G513 of HadA, hence opening up the possibility of applying this engineering approach to improving their thermostabilities as well. Molecular dynamics (MD) simulations confirmed that the improved thermostability of the G513Y variant was due to aromatic hydrocarbon interactions between Y513 and N359, L347, G348, and F349.     

Treatment of biodiesel wastewater by indirect electrooxidation: Effect of additives and process kinetics

Due to the presence of growth inhibitor and high impurity concentration in biodiesel wastewater, both biologicaln and chemical processes are ineffective for treating such wastewater. In this work, biodiesel wastewater was treated by electrooxidation via Ti/RuO2 electrodes in batch and continuous modes. Effects of the additive type, hydrogen peroxide (H₂O₂) and sodium chloride (NaCl), and concentration on the treatment efficiency, monitored in terms of the reduction in the biological oxygen demand (BOD), chemical oxygen demand (COD) and oil and grease level, were explored. The addition of NaCl gave higher treatment efficiency than H₂O₂, and both were higher than no addition, due to the continuous generation of the oxidizing chloride species. The removal of almost all the COD and oil and grease and ~95% BOD was obtained in the presence of 0.061 M NaCl at an applied current density of 4.28 mA/cm² for 7h. In continuous operation mode, the steady state condition was reached within 11 h and the treatment efficiency decreased as the wastewater feed rate increased. By using wastewater feed rate of 2mL/min, approximately 83.56, 61.43 and 91.72% of BOD, COD and oil and grease levels were respectively removed. The rate of pollutant removal fitted a first order reaction for both the batch and continuous operation modes.