Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.01.14.632876

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

Vallejos-Baccelliere, G.·Kaufman, S. B.
View original source ↗Export metadata ↓

Abstract

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

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Vallejos-Baccelliere, G., Kaufman, S. B.. 2025-01-16. Unveiling the influence of salt concentration on the different stages of the catalytic cycle of a halophilic enzyme. https://doi.org/10.1101/2025.01.14.632876

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

MgATP/MgADP-dependent conformational dynamics and intrinsically disordered regions of vascular KATP channels revealed by cryoEM

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

biochemistry↗
bioRxiv2026

Probing the sequence variability tolerance in a de novo α-helical barrel biocatalyst

De novo-designed enzymes have recently achieved high catalytic activity and stereoselectivity while demonstrating exceptional thermostability in entirely novel protein scaffolds. Among these, -helical barrel protein scaffolds are attractive structures for biocatalysis due to their structural simplicity, high thermostability, and rationalizable sequence patterning. However, enabling major structural reengineering of these scaffolds while maintaining the structure, stability and catalytic activity while also improving soluble protein production remain major challenges and pose the fundamental question how engineerable a de novo backbone-sequence pair is. Here, we combine deep learning based and classic computational protein design to modify and optimize de novo -helical barrel biocatalysts. Using the previously reported six-helical barrel 6H5L as a model scaffold, AlphaFold2-guided RosettaRemodel enabled the design of a truncated variant, whose crystal structure closely matches the design model. Additional sequence-redesign using ProteinMPNN generated a variant with a tenfold increase of soluble protein yield in Escherichia coli. Biochemical, biophysical, and structural analyses showed that both variants retained the overall barrel architecture, high thermal stability, and catalytic activity for both purified protein and whole-cell systems. Detailed kinetic analysis on the variants showed both variation in kcat and Km, reflecting changes in catalytic turnover and substrate binding. Together, these approaches provide new insights and possibilities for the further engineering of functional de novo -helical barrels, their ability to withstand dramatically large sequence changes and their broader application in biocatalysis and biotechnology.

biochemistry↗
bioRxiv2026

Cytokine-induced nuclear translocation of STAT1 via a non-transferable NLS

The targeting function of nuclear localization signals (NLSs) is generally considered independent of a protein's native sequence or fold and is readily transferable to heterologous cargos. Contrary to this paradigm, rapid nuclear translocation of phosphorylated STAT1 (pSTAT1) following cytokine stimulation requires importin {beta}, Ran-GTP, and the importin 5 isoform, which recognizes a non-transferable NLS. Here, we present cryo-EM structures of pSTAT1 bound to importin 5, revealing an asymmetric 2:1 complex that diverges from canonical NLS-mediated cargo recognition. Importin 5 occupies the DNA-binding groove of the pSTAT1 dimer, with a single STAT1 N-terminal domain positioning the C-terminal Armadillo repeats 9-10 (S1B domain) orthogonal to the DNA-binding interface. This interface is also targeted by the Ebola virus protein VP24, an antagonist of interferon signaling. We further show that Ran-GTP alone is insufficient to trigger nuclear release of pSTAT1, which additionally requires the exportin CAS. A cryo-EM reconstruction of the CAS-Ran-GTP-5 complex, supported by in vitro competition assays, demonstrates that CAS and pSTAT1 are mutually exclusive ligands for importin 5. Together, these findings define the molecular choreography of cytokine-induced STAT1 nuclear translocation and release, establishing a general paradigm for STAT family signaling.

biochemistry↗