Biochimie

The catalytic rate constant (kcat) for product formation is considered a turnover number. Therefore, it is often mistakenly believed that kcat equals the turnover number and the number of substrate molecules changed per unit of time. Therefore, the aim of this study is to show that the rate constant for product synthesis and release is not always the same as the rate constant [Formula] for substrate utilization. To determine the precise substrate concentration at which these two rate constants are identical, it is appropriate to derive equations that allow the computation of [Formula]. In the end, the study will provide the most likely concentration of enzymes that can guarantee minimal or no recycling. An analysis of the literature on invertase (EC 3.2.1.26) and the Bernfeld method of generating Michaelian kinetic parameters for human salivary alpha-amylase (HSAA, EC 3.2.1.1) revealed that all kinetic parameters except [Formula] increased with substrate concentration. Meanwhile, the values for invertase decreased from 0.0697 to 0.0361/min, and the values for HSAA decreased from 5,802.4687 to 3,213.0124/min. The magnitude of [Formula] for each substrate concentration ([ST]) is not always equal, except when [ST] is determined post-assay by computation or extrapolation. The lower [ST] at which [Formula] and kcat for [HSAA] are equal is 3.667540128 g/L (5.682584642 M), which is similar to the molarity of HSAA (5.6101967709 M). The kcat for HSAA was 11,930.9885/min. Future assays should aim to generate large amounts of data for a robust statistical analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/728646v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@c49b65org.highwire.dtl.DTLVardef@1b60655org.highwire.dtl.DTLVardef@159ba67org.highwire.dtl.DTLVardef@1dce0af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Natural product synthesis by non-ribosomal peptide synthetases (NRPS) is greatly defined by the substrate selectivity of the adenylation (A) domains. Previous assays for specificity determination were mainly performed in vitro and were requiring protein purification. In this work, we developed - based on NRPS engineering - a novel in vivo assay suitable for high-throughput application named ASCR (A domain screening). Using the recently described XUT fusion sites, A domains and their upstream condensation domains were assembled as di-domains to characterized NRPS model system, which allowed detection of defined tripeptide products via mass spectrometry directly after cell culture extraction. We evaluated the assay by screening in total 54 A domains from five known and seven uncharacterized NRPS, covering a broad range organism taxonomy and GC content of the investigated NRPS-encoding genes. Additionally, we applied the assay to elucidate and confirm the structures of novel cyclic pentapeptides derived from three novel NRPS from Photorhabdus temperata K122.

The chemokine-like peptide GPR15LG is a known agonist of G protein-coupled receptor 15 (GPR15), a ligand-receptor pair primarily implicated in mammalian mucosal immunity and lymphocyte homing. However, the evolutionary origin and phylogenetic distribution of this signaling system remain poorly understood due to the extreme sequence diversity of GPR15LG orthologs. In this study, we identified GPR15LG orthologs in several fish species for the first time according to their conserved gene synteny, genomic organization, and amino acid sequence features. A representative ortholog from the spotted gar (Lepisosteus oculatus), termed Lo-GPR15LG, was recombinantly prepared and functionally characterized using NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay and homogenous ligand-receptor binding assay. Our results demonstrated that Lo-GPR15LG directly binds to and efficiently activates its cognate receptor, Lo-GPR15, with a dissociation constant (Kd) of approximately 60 nM and an EC50 value of approximately 10 nM. Functional assays further revealed that receptor activation is critically dependent on the conserved C-terminal residues. Notably, human and fish orthologs exhibited no cross-species activity, consistent with their high sequence divergence. These findings reveal that the GPR15LG-GPR15 signaling system originated in ancient fish ancestors and has remained a conserved signaling axis throughout vertebrate evolution, suggesting a fundamental role in immunity across all vertebrate lineages.

Glucose has important roles in the development of the hematopoietic stem cells and the brain in vertebrate embryos; however, in most oviparous animals, the amount of glucose in the yolk is scarce. In zebrafish, gluconeogenesis takes place in the yolk syncytial layer (YSL), an extraembryonic tissue that surrounds the yolk. Gluconeogenic activity have also been observed in extraembryonic YSL-like tissue or endoderm-derived tissues in cloudy catshark, sterlet, and western clawed frog during development. However, it remains unclear when such ability was acquired or how it changed over the evolution of vertebrates. In this study, we used the Arctic lamprey, a cyclostome sister group of jawed vertebrates, to compare changes in metabolite levels and gluconeogenic gene expression patterns during development. Also, gluconeogenic activity was assessed using 13C-labeled substrates. Our metabolite analysis revealed that glucose levels increased during development and that glycerol was actively metabolized to produce glucose. In addition, many gluconeogenic genes were expressed in the muscle, notochord, and epithelium, making a striking contrast to previous observations in the above-mentioned vertebrates. Genomic DNA sequence motif analysis using HOMER and MEME identified common transcription factors binding motifs in the upstream regions of g6pc1/2 and fbp1 across vertebrate lineages. Among them, interestingly, the binding motif for HNF4A was not detected in g6pc1/2 and fbp1 genes of cyclostomes, suggesting distinct transcriptional regulation of gluconeogenesis in cyclostomes. These results indicate that gluconeogenesis is an essential process during development across vertebrate lineages, including cyclostomes, although the tissues and regulatory mechanisms for this function vary among lineages.

Transcriptional termination efficiency is considered an important parameter for fine tuning bacterial gene expression. Still, the design principles that determine transcription termination efficiency remain poorly understood. In this study, we aimed to investigate the impact of the 3' untranslated region (3'UTR) on gene expression in Escherichia coli and other bacteria. First, 3'UTR variant sequences were generated, with randomized 30 bp sequences inserted between the STOP-codon and an intrinsic terminator, consisting of a GC-rich hairpin and a downstream poly(U)-tail. Using three reporter genes, it was found that different 3'UTR sequences resulted in an up to five-fold difference in protein production, independent of the upstream coding sequence. The highest protein production was achieved when an adenosine was present directly upstream of the terminator hairpin. This was consolidated by systematic substitution of key nucleotides of the terminator and assessing their effect on mRNA and protein levels. Subsequently, we developed a predictive random forest machine learning model trained on the termination efficiency of different natural and synthetic terminator sequences, revealing an important role for the nucleotides directly upstream of the terminator hairpin. Altogether, this study showed that an additional adenosine nucleotide upstream of the terminator hairpin leads to improved protein production while reducing terminator read-through.

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

We have developed a fluorometric assay for detecting reductase activity in biological samples through 4-methoxy-1-naphthalenemethanol (MONOL-41) formation. The enzyme carbonyl reductase 1 (CBR1) and four members of the aldo-keto reductase (AKR) 1 family (AKR1A1, AKR1B1, AKR1B10, AKR1C3) were evaluated for their ability to reduce 4-methoxy-1-naphthaldehyde (MONAL-41). AKR1B1 and CBR1 followed Michaelis-Menten kinetics, whereas AKR1B10, AKR1A1, and AKR1C3 showed substrate inhibition above 10 {micro}M (70 {micro}M for AKR1C3). Among the tested enzymes, AKR1B10 displayed the highest catalytic efficiency in the absence of substrate inhibition. The MONOL-41 assay was compared with the standard NADPH-based method, showing improved sensitivity, robustness, and lower detection limits (0.77 {micro}g/mL vs. 1.49 {micro}g/mL). These results confirm its suitability for monitoring AKR1B10 activity. The assay was then applied to A549 cell extracts, which express multiple reductases. Activity decreased at substrate concentrations above 10 {micro}M, suggesting a predominant role of AKR1B10. Inhibition studies using tolrestat and high MONAL-41 concentrations indicated a limited contribution of CBR1 ([~]7-8%). Considering both catalytic efficiency and expression levels, AKR1B10 appears to be the main contributor to reductase activity in this model. In A549 living cells, MONAL-41 showed no cytotoxicity up to 50 {micro}M and enabled real-time monitoring due to its membrane permeability. However, oxidation by aldehyde dehydrogenases can generate MONOIC-41, which has similar spectral properties but a lower quantum yield, potentially affecting signal interpretation. Overall, this assay represents a sensitive and cost-effective tool for detecting reductase activity and screening inhibitors.

Sickle cell disease (SCD) is caused by a single amino acid substitution in the {beta}S globin chain at 6th position (6E[->]V). This results in polymerization of deoxy state of sickle hemoglobin (HbS), followed by its precipitation and subsequent sickling of erythrocytes. These deformed cells can block small capillaries (vaso-occlusion), causing cardiovascular complications, ultimately leading to ischemia-reperfusion injury, severe oxygen deficiency, and progressive systemic damage. Occasionally, patients with SCD have been observed to produce exorbitantly high levels of fetal hemoglobin (HbF), which has been linked with the inhibition of HbS polymerization. One of the effects of hydroxyurea, the most commonly used therapeutic for SCD, is to elevate HbF levels. However, the mechanism of inhibitory role of HbF on HbS polymerization is largely unknown. This study attempts to gain insights into the mechanisms involved in this process by means of native mass spectrometry, ion mobility mass spectrometry, and hydrogen deuterium exchange-based mass spectrometry (H/DX-MS). The conformational flexibility of asymmetric hemoglobin, HbFS (2{gamma}{beta}S), for the observed regions in the tetrameric molecule appears to be more in the deoxy state as compared to the oxy state, eventually leading to reduced polymerization of sickle hemoglobin in patients with SCD that express elevated HbF levels.

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduction in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability assays. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold increase in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.

Prenylated isoflavonoids are widely distributed specialized metabolites within the Fabaceae and contribute to various characteristic biological activities for both plants and humans. Several aromatic prenyltransferases (PTs) have been identified in Glycyrrhiza species, which are the most widely consumed crude drugs in traditional Chinese medicine. However, these enzymes do not sufficiently explain the structural diversity of prenylated flavonoids produced in the Glycyrrhiza genus. To identify additional novel PTs, we used elicited cultured Glycyrrhiza glabra roots as source material, in which elicitor treatment of cultured roots increased the accumulation of multiple prenylated flavonoids. To identify the responsible enzyme, PT candidates were screened using G. uralensis transcriptomes, currently the sole publicly available transcriptomic resource within the genus, and a homolog designated GgBSPT1 (BSPT; a broad-substrate prenyltransferase) was subsequently isolated from elicited cultured G. glabra roots. GgBSPT1 differed from previously identified Glycyrrhiza PTs in both amino acid sequence and enzymatic properties. GgBSPT1 catalyzed 3'-prenylation of isoliquiritigenin and 6-prenylation of five flavonoids, i.e., this PT displayed broad substrate acceptance across 20 distinct flavonoid structures. Overall, elicited cultured G. glabra roots enabled the identification of a previously unrecognized PT that is functionally distinct from earlier reported Glycyrrhiza PTs. This study provides a new insight into the metabolic plasticity of Glycyrrhiza species and expands the enzymatic toolkit for future metabolic engineering of prenylated phytochemicals by the unusually broad substrate specificity of GgBSPT1. Main conclusionUsing cultured Glycyrrhiza glabra roots, we identified a new prenyltransferase involved in the formation of a variety of flavonoids, thereby revealing novel prenylated isoflavonoid pathways in licorice.

In many oviparous animals, egg yolk is the sole source of nutrition until feeding begins, and carbohydrates are present in only small amounts in the yolk. Glucose plays an important role in the developmental processes of various animals. In addition, gluconeogenesis has been reported to occur in the yolk syncytial layer (YSL) of cartilaginous fish and teleosts. In contrast, the role of gluconeogenesis in tetrapods remains unclear. In this study, we used Xenopus tropicalis, an anuran amphibian, which lacks YSL, and therefore provide an opportunity to examine the evolutionary conservation of gluconeogenic mechanisms among vertebrates. In X. tropicalis, liquid chromatography/mass spectrometry revealed that glucose levels increased before liver formation. Subsequent tracer experiments using 13C-labeled metabolic substrates detected gluconeogenesis activity from glycerol and lactate. Expression analyses showed that gluconeogenic genes are expressed in the epidermis and endoderm. Consistently, G0 knockout of fbp1, a key gluconeogenic gene, resulted in a significant reduction in glucose levels, affecting brain development. These findings first demonstrate that gluconeogenesis supports development of X. tropicalis. To the best of our knowledge, gluconeogenesis in developing epidermis has not been reported, highlighting previously unrecognized diversity in tissue-specific metabolism during vertebrate development. Comparative analyses across species will provide further insights into the evolution and functional significance of embryonic gluconeogenesis and nutrient metabolism.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Rhodococcus opacus is an oleaginous actinobacterium with considerable potential for lipid-based bioproduction, as well as for utilising a variety of carbon sources as substrates, including renewable, cost-effective resources. Although its capacity for triacylglycerol accumulation is well established, the regulatory logic that governs its fatty acid and mycolic acid biosynthesis is still poorly understood. Here, we investigated the transcriptional control of the type II fatty acid synthase (FASII) pathway in R. opacus PD630, revealing a regulatory architecture that is more complex than previously assumed. Differential gene expression analysis showed that environmental cues, including temperature, pH, carbon-to-nitrogen ratio and the presence of free fatty acids influence the FASII gene cluster expression in a non-uniform manner. This phenomenon suggests the presence of internal transcription start sites and modular regulation within the cluster. We identified three lipid-responsive transcription factors, MabRRO, FadR1RO and FadR2RO, that are all capable of binding the fasII promoter in vitro. DNA binding of FadR1RO and FadR2RO was disrupted by long-chain acyl-CoA molecules, indicating ligand-dependent control. Together, these findings reveal previously unrecognised layers of transcriptional regulation in the R. opacus FASII pathway and highlight both conserved and divergent regulatory features within the Mycobacteriales lineage. Featured Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/678588v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@456a6dorg.highwire.dtl.DTLVardef@14e3d1forg.highwire.dtl.DTLVardef@18ed368org.highwire.dtl.DTLVardef@1d979a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Marine macroalgae are valuable sources of bioactive compounds. In this study, we thus investigated the chemical composition and biological activity of an extract from the green seaweed Ulva lacinulata, composed of small bioactive compounds. Comprehensive compositional analyses and high-resolution mass spectrometry revealed its diverse molecular profile composed in particular of peptides/amino acid derivatives, saccharides, low-chain fatty diacids, oxylipins and minerals. Its anti-inflammatory activity was assessed after 6 h pre-treatment in LPS-stimulated cultured RAW 264.7 macrophages, showing that it significantly and dose-dependently reduced the expression and/or secretion of pro-inflammatory cytokines such as TNF-alpha; and IL-6, and targeted the NF-kB signaling cascade. It modulated the SIRT1-AMPK signaling axis and increased the LC3-II/LC3-I ratio, supporting the activation of a controlled autophagic response. This work highlighted the potential of this marine-derived extract as a safe and effective functional ingredient for the development of functional food and/or dietary supplements targeting chronic low-grade inflammation.

Cholera, caused by Vibrio cholerae, continues to pose a serious global public health challenge, with its impact worsened by rising antibiotic resistance associated with bacterial biofilm formation. This study reveals the role of the hypothetical protein (HP) TYC33605.1 in cyclic-di-GMP (c-di-GMP)-mediated biofilm regulation and identifies natural inhibitors that disrupt this mechanism. Functional annotation revealed TYC33605.1 as a membrane-associated diguanylate cyclase (DGC) with GGDEF and sensory domains, critical for c-di-GMP synthesis and biofilm persistence. Homology modelling and molecular dynamics (MD) simulations validated its stable 3D structure (C-score: -1.22, Ramachandran favoured regions: 91.1%) and dynamic behaviour (average RMSD: 8.55 [A]). Virtual molecular docking screening of 1,092 natural compounds identified Luteolin (CID 5280445) and Sativanone (CID 13886678) as top candidates, exhibiting strong binding affinities (-9.1 and -9.0 kcal/mol, respectively) and forming hydrogen bonds, {pi}-cation, and hydrophobic interactions with key residues (Glu293, Arg364, Ala176). MD simulations (100 ns) confirmed complex stability, with Luteolin and Sativanone showing lower RMSD fluctuations (7.78 [A] and 8.13 [A]) compared to the control and apoprotein. The ADME/Tox profiles highlighted favourable pharmacodynamics (PD), pharmacokinetics (PK), high gastrointestinal absorption, no hepatotoxicity, and drug-likeness (Lipinski compliance). Principal component, probability density function, and binding free energy analyses underscore ligand-induced conformational stability. This study proposes the molecular characterisation of the HP and the bioactive compounds Luteolin and Sativanone as promising inhibitors targeting TYC33605.1, offering a novel strategy to combat biofilm-mediated antibiotic resistance and a framework for analogous antimicrobial discovery in Vibrio cholerae.

Resistance to {beta}-lactam antibiotics, including carbapenems, mediated by metallo-{beta}-lactamases (MBLs), including the New Delhi metallo-{beta}-lactamase (NDM) MBL subfamily, is increasing. No MBL inhibitors are currently approved for clinical use with most reported MBL inhibitors are metal ion chelators, acting either at the Zn(II) ion active site and/or in solution. The hexokinase inhibitor 3-bromopyruvate (3-BP) is reported to inhibit NDM-1. We found that 3-BP selectively restored the antimicrobial activity of meropenem against carbapenem resistant Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii strains, obtained from clinical and environmental isolates from Tanzania and Malawi, containing genes that encode NDM-1 or NDM-5, but not against strains containing genes encoding for serine {beta}-lactamases. Mass spectrometry studies with NDM-1 and NDM-5 support a mechanism involving covalent reaction of 3-BP with an active site cysteine residue. The results will promote work on the development of covalently reacting MBL inhibitors, a strategy that has been successful for inhibition of the nucleophilic serine {beta}-lactamases.

Legumain (AEP) is a cysteine protease with a highly conserved catalytic core but variable surface features whose evolutionary and structural diversity remain incompletely understood. To investigate how these features differ across vertebrates, we compared human legumain with six shark orthologs using sequence alignment, AlphaFold based structural modeling, pocket detection, and qualitative docking. All shark sequences retained the canonical His-Cys dyad and {beta} sandwich fold, and structural superposition revealed strong global conservation (RMSD = 0.39 [A]). A single surface exposed loop adjacent to a shallow cavity--designated Pocket 2--displayed pronounced sequence divergence. Structural models reproduced this pattern, with Pocket 2 showing the greatest variation in geometry and residue composition across species, including Callorhinchus milii, which clustered with elasmobranchs in Pocket 2 features. Although loop conformations were predicted with lower confidence, Pocket 2 was consistently detected in all models and exhibited interspecific differences in volume, shape, and physicochemical environment. Docking of a structurally characterized reference ligand (5KN) into Pocket 2 revealed species specific differences in modeled binding orientations and interaction patterns that were consistent with these geometric variations, though not interpretable as quantitative affinity predictions. Together, these results identify Pocket 2 as a structurally conserved but locally variable region of legumain and highlight it as a candidate site for future experimental and computational investigation. This study is hypothesis generating and provides a framework for examining how localized structural variation may arise within an otherwise conserved protease family.