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Kaufman, S. B.

Publications and source records attributed to Kaufman, S. B..

2 recordsLinked to original sources
bioRxiv2025

Unveiling the influence of salt concentration on the different stages of the catalytic cycle of a halophilic enzyme

Enzymes from halophilic organisms have adapted to function in salt concentrations near saturation, making them an interesting model for studying the effect of salt on enzyme catalysis. The main insights into the effect of ionic strength on enzyme catalysis come primarily from enzymes with positively charged surfaces interacting with negatively charged substrates (e.g., ribonucleases), whose activity decreases at high salt concentrations. In this study, we investigated the effect of salt on the kinetics of Glucose-6-phosphate dehydrogenase (G6PDH) from the halophilic archaeon Haloferax volcanii (HvG6PDH), which has optimal activity conditions at concentrations exceeding 2 M KCl. The enzyme catalyzes the NAD+-dependent oxidation of G6P, a negatively charged substrate, and glucose, a non-charged substrate. Using steady-state kinetics, we determined that the enzyme follows an ordered-sequential kinetic mechanism, with NAD+ being the first substrate to bind and NADH being the last released product. Through steady-state kinetic experiments, we found that the main effect of salt is on the KM for G6P, which decreased approximately 50-fold. For glucose dehydrogenase (glcDH) activity, the main effects were a 10-fold increase in kcat and a roughly 10-fold increase in kcat/KM for glucose. To analyze the effect of salt on the different stages of the catalytic cycle, we performed pre-steady-state experiments for both activities. We found that KCl did not affect the catalytic step in G6PDH activity, but it did increase the rate of catalysis in glcDH. Using a minimal model that accounts for substrate binding, chemical transformation, and product release, we determined that the main effect on G6PDH activity was an increase in the rate of G6P association. In contrast, for glcDH activity, the main effect was an increase in the rates of catalysis and product release. The results show that charge screening plays an essential role in the effect of salt on catalysis. Furthermore, it suggests differences in ion penetration to the active site between the two activities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/632876v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@782511org.highwire.dtl.DTLVardef@1c9bce1org.highwire.dtl.DTLVardef@18f63faorg.highwire.dtl.DTLVardef@ecd1a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2022

A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers

The fijivirus Mal de Rio Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric {Delta}C-arm version. In addition, although both P9-1 and P9-1{Delta}C-arm catalyze the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between the ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Rio Cuarto crop disease.

biochemistry↗