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Background: DJ-1 is a protein whose mutation causes rare heritable forms of Parkinson’s disease (PD) and is of interest as a target for treating PD and other disorders. This work used high performance affinity microcolumns to screen and examine the binding of small molecules to DJ-1, as could be used to develop new therapeutics or to study the role of DJ-1 in PD. Non-covalent entrapment was used to place microgram quantities of DJ-1 in an unmodified form within microcolumns, which were then used in multiple studies to analyze binding by model compounds and possible drug candidates to DJ-1. Results: Several factors were examined in optimizing the entrapment method, including the addition of a reducing agent to maintain a reduced active site cysteine residue in DJ-1, the concentration of DJ-1 employed, and the entrapment times. Isatin was used as a known binding agent (dissociation constant, ~2.0 µM) and probe for DJ-1 activity. This compound gave good retention on 2.0 cm × 2.1 mm inner diameter DJ-1 microcolumns made under the final entrapment conditions, with a typical retention factor of 14 and elution in ~8 min at 0.50 mL/min. These DJ-1 microcolumns were used to evaluate the binding of small molecules that were selected in silico to bind or not to bind DJ-1. A compound predicted to have good binding with DJ-1 gave a retention factor of 122, an elution time of ~15 min at 0.50 mL/min, and an estimated dissociation constant for this protein of 0.5 µM. Significance: These chromatographic tools can be used in future work to screen additional possible binding agents for DJ-1 or adapted for examining drug candidates for other proteins. This work represents the first time protein entrapment has been deployed with DJ-1, and it is the first experimental confirmation of binding to DJ-1 by a small lead compound selected in silico. 

Abstract Plants release back to the atmosphere about half of the CO 2 they capture by photosynthesis. Decreasing the rate of crop respiration could therefore potentially increase yields, store more carbon in the soil and draw down atmospheric CO 2 . However, decreasing respiration rate has had very little research effort compared to increasing photosynthesis, the historically dominant metabolic paradigm for crop improvement. Conceptual and technical advances, particularly in protein turnover and directed enzyme evolution, have now opened ways to trim the large fraction of respiration that fuels proteome maintenance by lowering the breakdown and resynthesis rates of enzymes and other proteins. In addition to being theoretically possible and practicable, exploring the reduction of respiration is prudential, given that it (i) has barely yet been tried and (ii) could help meet the challenges of sustaining crop productivity and managing atmospheric carbon. 

Abstract These studies reveal the first structure ofClostridium acetobutylicumalcohol dehydrogenase (CaADH), a protein exhibiting remarkable substrate promiscuity and stereochemical fidelity. The CaADH enzyme is utilized here for synthesizing 20 potential aryl isoserine side chains for the Taxotere family of tubulin‐binding chemotherapeutics. The approach involves dynamic reductive kinetic resolution (DYRKR) upon the corresponding α‐chloro‐β‐keto esters, showing high D‐synstereoselectivity, including those leading to the clinically relevant milataxel (Ar = 2‐furyl) and simotaxel (Ar = 2‐thienyl) side chains. Furthermore, various cross‐coupling chemistries performed on thep‐bromophenyl isoserine side chain significantly enhance the structural diversity of the taxoid side chain library obtained (16 additional taxoid side chains). The CaADH structure is notable: (i) the nicotinamide cofactor is bound in ananti‐conformation, with the amide carbonyl occupying the ketone binding pocket, and (ii) a flexible loop near the active site likely contributes to the remarkable substrate promiscuity observed in CaADH. We present our perspective on the dynamic nature of the CaADH active site through molecular dynamics simulation, proposing a halogen bonding model as a potential mechanism for the remarkable selectivity for an (S)‐configured C─Cl bond, in addition to the D‐facial selectivity, demonstrated across 20 diverse substrates by this remarkable short‐chain dehydrogenase enzyme.