Biochemistry

A recent genome mining study identified class II lanthipeptides encoded in Nostoc punctiforme PCC73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were not determined. In this study, we heterologously produced the nostolysamides by co- expression of the NpuA precursor peptide with an N-terminal SUMO tag with the class II lanthipeptide synthetase NpuM in Escherichia coli. We structurally characterized the NpuA-derived product and established the position of the thioether crosslinks. All four lanthionine and methyllanthionine residues were shown to have the DL configuration by Marfeys analysis. Tandem mass spectrometry as well as mutagenesis studies indicate an N-terminal non-overlapping methyllanthionine ring and three overlapping rings at the C-terminus for which the most likely ring pattern is proposed. After removal of the leader peptide, the resulting lanthipeptide exhibits antifungal activity against Candida species as well as antimicrobial activity against gram positive bacteria by disrupting cell membranes. The antibacterial activity is shown not to involve lipid II, consistent with the observed antifungal activity because fungi do not contain this bacterial cell wall precursor. The biosynthetic gene cluster also encodes an acetyltransferase NpuN that transfers long chain acyl groups to the side chain of a Lys residue in position 1 of the precursor peptide. In vitro studies of NpuN shows relatively broad substrate specificity with NpuN conjugating various acyl groups from acyl-CoA substrates to Lys1 in the nostolysamides. The acylation did not appreciably change the antifungal and antimicrobial activity of nostolysamide showing that it is not required for these activities.

SNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipids impact fusion through direct protein-lipid interactions or through modulating the physical properties of membranes to affect protein function. Lysophospholipids (LPLs) can affect membrane curvature, fluidity and energy of deformation. Their effects are due to their head group, and the length and saturation of their single acyl chains. Here we examined how the properties of LPLs affect yeast vacuole fusion and ion transport. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons inhibited fusion with IC50 values of {cong} 40-120 {micro}M. While acyl chain length moderately affected fusion, the head group played a major role. Unlike LPCs, Lysophosphatidic acid (LPA 18:1) failed to fully inhibit fusion, while lysophosphatidylethanolamine (LPE 18:1) had no effect. Separately we found that changes in acyl chain length and saturation differentially affected Ca2+ transport and vacuole acidification. Together these data show that the effects of LPLs on membrane fusion and ion transport were due to a combination of head group type and acyl chain length.

Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin (Erithacus rubecula) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a (ErCRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing ErCRY4a with the fbIC gene that encodes for 8-HDF synthase and thus for production of 8-HDF in E. coli. As a positive control for 8-HDF binding, we expressed Xenopus laevis 6-4 photolyase (Xl6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl6-4PL, but not to ErCRY4a. We studied the binding of MTHF using in vitro reconstitution followed by UV-Vis spectroscopy and isothermal titration calorimetry (ITC) assays. No interaction was observed between MTHF and ErCRY4a. To theoretically understand the binding of potential antenna chromophores to ErCRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of ErCRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that ErCRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Cellular transport processes along microtubules are often facilitated by multi-motor complexes, which are connected by adapter proteins and cargoes. The nuclear pore protein Nup358, for example, interacts with the dynein adapter Bicaudal D2 (BicD2), which in turn recruits minus-end directed dynein motors and plus-end directed kinesin-1 motors for a nuclear positioning pathway that is essential for brain development. How motor recruitment is regulated by interactions of BicD2 with Nup358 is not well understood. Here, we characterize the structure of a minimal complex of kinesin-1 light chain 2 (KLC2), Nup358 and BicD2 by cryo-electron microscopy and small angle X-ray scattering. KLC2/Nup358 assumes a rod-like structure that increases in thickness, when BicD2 is bound. The addition of BicD2 also shifts the KLC2/Nup358/BicD2 complex towards a 2:2:2 stoichiometry, promoting dimerization at lower protein concentrations than without BicD2. Similarly, the presence of the Nup358/KLC2 interaction results in a shift towards a 2:2:2 stoichiometry. Based on these results, we hypothesize that KLC2 and BicD2 are recruited to Nup358 in a cooperative manner, and cooperativity may be promoted by modulation of the oligomeric state.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) essential for inhibitory synaptic transmission throughout the central nervous system. Despite progress in understanding their three-dimensional structure, the molecular basis for how neurotransmitter binding is transduced to ion channel gating remains poorly understood. Furthermore, relatively little is known about the contributions of distinct subunits to this coupling within typical heteromeric receptors. A highly conserved proline (site 1) in the M2-M3 linker of pLGIC subunits is involved in channel gating - e.g., P273 in the GABAAR {beta}2 subunit. In GABAARs, only the {beta} subunits have an additional proline in the M2-M3 linker (site 2) - e.g., {beta}2(P276) - whereas all other subunits have a non-proline at the homologous site 2 position. Here, we investigate the functional contribution of proline at site 2 in distinct subunits of 1{beta}2{gamma}2 GABAARs. We expressed wild type or mutant 1{beta}2{gamma}2 GABAARs in Xenopus laevis oocytes and used two-electrode voltage clamp electrophysiology to record channel currents in response to GABA and/or other ligands. First, we introduced a proline at site 2 in 1 or {gamma}2 subunits: 1(A280P) and {gamma}2(S291P). Second, we replaced the site 2 proline in the {beta}2 subunit with its homologous non-proline residue from 1 or {gamma}2 subunits: {beta}2(P276A) or {beta}2(P276S). We show that 1(A280P) confers enhanced GABA-sensitivity and spontaneous unliganded channel activity, whereas {gamma}2(S291P) has minor effects on channel activation. In contrast, {beta}2(P276A) or {beta}2(P276S) either had no effect or enhanced GABA-activation, respectively, indicating complex functional dependence on the side chain at site 2 in the {beta}2 subunit. When in combination with other substitutions, the presence or absence of 1(A280P) was consistently correlated with enhanced GABA-sensitivity and spontaneous activity. Thus, introduction of a proline at site 2 in the 1 M2-M3 linker biases the channel towards an activated state and prevents it from remaining closed at rest.

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. Here, we use site-directed mutagenesis to study the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Mutations at this position change the biochemical properties (KM for CO, product inhibition constant, catalytic bias...), and increase the resistance of the enzyme to the inhibitor O2, showing that isoleucine 563 indeed lines the gas channel. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far. We show that the size of the introduced amino acids is less important than their flexibility - along with the size of the cavity formed near the active site in the channel. We also conclude that O2 access to the active site cannot be slowed down without also affecting CO diffusion. This tradeoff will have to be considered in further attempts to use site-directed mutagenesis to make CODHs more O2 tolerant.

Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on its release from a precursor protein through peptide bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we describe the in vitro reconstitution of cholesterolysis activity for SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multi-well plates by monitoring changes in fluorescence resonance energy transfer (FRET) from an engineered precursor construct, expressed in E. coli and purified in soluble form. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol, (KM, 1-2 {micro}M, cholesterolysis t1/2 of [~]11 min) while rejecting the 3-alpha epimer, epi-cholesterol (KM > 100 {micro}M, t1/2 > 10 hr). By screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we identify cationic detergents that inhibit cholesterolysis and find a shared preference for the zwitterionic n-dodecyl-phosphocholine (DPC, Fos-choline-12), which supported the fastest reaction kinetics. Lastly, we report that alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC blocks cholesterolysis; however, activity could be chemically rescued with rationally designed hyper-nucleophilic sterols. Of those sterols, 2-beta carboxy cholestanol was active as a substrate with D46A variants only; the remaining sterols were accepted by both D46A and wild-type SHhC. In summary, we have established the first in vitro kinetic assay to continuously monitor enzymatic activity of wild-type and mutant vertebrate SHhC domains in multi-well plates, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis in vivo.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

MagLOV2 is an engineered flavoprotein designed to have large changes in fluorescence intensity in response to weak magnetic fields. Here, we characterize the magnitude of these fluorescence changes, known as the "magnetic field effect," as a function of the strength of an externally applied magnetic field in E. coli colonies expressing MagLOV2. We observe that the magnetic field effect is positive at low magnetic fields, reaches a maximum positive value near 1 mT, and then decreases, reversing sign at approximately 2 mT. Furthermore, the effect starts to plateau above approximately 70 mT, with a decreased sensitivity of fluorescence changes to magnetic fields above this range. The non-monotonic behavior, as well as the diminished responsiveness to higher magnetic fields, are consistent with the changes in fluorescence being driven by electron spin-dependent chemical processes governed by the radical pair mechanism.

Under inflammatory conditions, the ubiquitin-like modifier FAT10 serves as a tag for protein degradation by the 26S proteasome. FAT10 is degraded along with its substrates and this process is independent of the segregase VCP/p97, which, in the regular ubiquitin pathway of degradation, is required if a substrate lacks a disordered initiation region. FAT10 itself is loosely folded and its tendency to aggregate has complicated investigations of its structure, interaction, and function. Recently hydrogen-deuterium exchange in combination with mass spectrometry has suggested that, in preparation of degradation by the proteasome, the adapter protein NUB1 traps FAT10 in a mostly unfolded state by capturing a {beta}-strand. {beta}-strand capture was subsequently confirmed by magic-angle spinning (MAS) NMR spectroscopy of a stabilized variant of the N-domain of FAT10 in complex with NUB1L, the longer splice variant of NUB1. MAS NMR, in addition, revealed that the N-domain of FAT10 and NUB1L form a fuzzy complex and that the N-terminus of FAT10 is positioned for initiation of degradation by specific non-covalent interaction with NUB1L. Here, we report the investigation of the wild-type N-domain of FAT10 by MAS NMR. Co-sedimentation with NUB1L yields high-quality spectra, which enable sequential assignment of resonances. Through the lens of MAS NMR, the complexes of the wild-type and stabilized N-domain of FAT10 with NUB1L are identical. The N-terminus of FAT10 again shows up prominently in the spectra, even though the residue is this time an Ala, not a Gly. Our experience suggests that co-sedimentation in combination with MAS NMR is generally helpful in the exploration of conditional folds of intrinsically disordered proteins.

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

Glutamate racemase (MurI) catalyzes the stereochemical interconversion of L-glutamate to D-glutamate, a key element of bacterial peptidoglycan biosynthesis. In this study, we present the crystal structure of Helicobacter pylori glutamate racemase at 1.43 [A] and in monoclinic symmetry, as previously reported models, but different unit-cell parameters. The present model contains a single dimer and retains the previously described head-to-head dimer arrangement. The differences between the models arise from variations in unit-cell parameters, which lead to altered crystal packing interactions rather than changes in the quaternary assembly. The monomeric fold and active-site architecture remain conserved and are consistent with the catalytic features described for bacterial glutamate racemases. This structure provides an updated, high-resolution structural model for H. pylori glutamate racemase and highlights the variability in crystal packing within the same space group.

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of Nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

The KRAS oncogene, central to cellular signaling via MAPK and PI3K-AKT pathways, is a notorious cancer driver frequently activated in pancreatic, colorectal, and lung carcinomas. Regulation of human KRAS oncogene expression is important due to its capital role in cell growth, proliferation, and survival. Misregulation of its expression contributes directly to the development and progression of multiple types of cancer. In previous studies, the role of G-quadruplexes elements in both the promoter and 5 UTR regions have shown to play important roles in KRAS expression, particularly when these G4s elements interact with regulatory protein hnRNPA1. In this study, we reveal that KRAS expression is also modulated at the post-transcriptional level through the formation of RNA G-quadruplexes (rG4s) situated at the 5 untranslated region (5UTR) of the mRNA. Biophysical and binding studies were carried out to probe the interaction. Through isothermal titration calorimetry (ITC), we quantified a strong binding affinity between the UP1 domain of hnRNPA1 and short-nucleotide RNA segments capable of adopting different G-quadruplex fold. The binding interaction is characterized by a favorable Gibbs free energy change in the range of {Delta}G {approx} -32 to -34 kJ/mol, suggesting a specific and energetically favorable association. One-dimensional and two-dimensional 1H-15N HSQC NMR spectroscopy revealed pronounced chemical shift changes in residues of both RNA recognition motifs (RRMs) of UP1, signifying direct contact with the rG4 structure.

The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO2 into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO2. In these strains, methanol oxidation is catalysed by an alcohol oxidase (Aox2), which transfers electrons to oxygen without conserving reducing equivalents. To address this limitation, in this study we explored redirecting methanol dissimilation through the native alcohol dehydrogenase (Adh2), coupling methanol oxidation with NADH generation to improve carbon efficiency. By deleting AOX2 and overexpressing ADH2, we generated Adh2-based autotrophic strains that exhibited growth rates comparable to the parental strain (0.007 h-{superscript 1}), while reducing specific CO2 production by 53% and increasing biomass yield (YX/MeOH) by 59%. We further applied this strategy to convert previously developed autotrophic strains producing itaconic acid and lactic acid into Adh2-dependent strains. Optimizing ADH2 expression through multicopy integration resulted in strains with approximately two-fold higher molar carbon efficiency (Y(X+P)/CO2) while achieving elevated product titers--2.2-fold for itaconic acid and 3.8-fold for lactic acid--relative to the parental strains. Our findings demonstrate that alcohol dehydrogenase-mediated methanol dissimilation can significantly improve yield and productivity of autotrophic K. phaffii strains, with broad implications for sustainable bioproduction from one-carbon substrates.

The European honey bee (Apis mellifera L.) is a key agricultural pollinator frequently exposed to pesticide residues, yet the molecular basis of its chemical adaptation, particularly glutathione S-transferases (GSTs) involved in xenobiotic detoxification, remain incompletely understood. In this study, AmGSTO1 was structurally and functionally characterized to evaluate its role in agrochemical interaction and protection against oxidative stress. The crystal structure of AmGSTO1 in complex with glutathione revealed its 3D architecture and key active-site residues were identified by structural analysis and site-directed mutagenesis. Fluorescence binding assays demonstrated measurable affinity for multiple agrochemicals, including TCP, fenoprop, 2,4-D, tetramethrin, nicotine, and 3-phenoxybenzaldehyde. However, HPLC analysis showed no detectable substrate depletion, suggesting ligand binding to AmGSTO1 without catalytic turnover. AmGSTO1 exhibited antioxidant activity toward cumene hydroperoxide, hydrogen peroxide, and paraquat, as well as dehydroascorbate reductase activity. These findings indicate that AmGSTO1 may contribute to agrochemical tolerance through ligand sequestration and redox protection mechanisms.

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

Disulfide bond formation is crucial to the structure and function of many proteins. It is known that there is diversity in the pathways for disulfide bond formation in bacteria and that there are gaps in our knowledge of these pathways. Using a combination of experimental and bioinformatic approaches we show that some of these gaps can be filled by a newly discovered oxidative folding pathway centered on methylamine utilization protein E (MauE). MauE has previously been associated with the methylamine utilization (MAU) gene cluster, which is involved in methylamine metabolism, in particular it is associated with the maturation of the small subunit of methylamine dehydrogenase. Here we show MauE from Caldithrix abyssi and Desulfatibacillum alphaticivorans functionally replace disulfide bond formation protein B (DsbB) in E. coli using two independent disulfide bond dependent assays. Furthermore, MauE is found in 14 species from 2 bacterial phyla that lack known pathways for structural disulfide bond formation, but which have proteins with structural disulfide bonds in the protein data bank. The active site for MauE was determined to be a conserved CXC motif. Using molecular docking predictions, we demonstrate that MauE is likely to interact with ubiquinone, similarly to the well characterized bacterial DsbB. We also constructed a dataset across thirty-five different phyla to demonstrate that MauE is potentially the second most common disulfide bond formation protein in bacterial disulfide bond formation pathways after DsbB. In addition, the distribution of MauE largely differs from the distribution of other MAU gene cluster markers affirming its role as a newly discovered generalist disulfide bond formation protein rather than being a specialized maturation factor for methylamine dehydrogenase. We also reveal further gaps in disulfide bond pathways, as well as species which may contain redundancies in their disulfide bond pathways.