Trypanosoma cruzi, the etiological agent of Chagas disease, depends on glycolysis for ATP production, rendering its glycolytic enzymes attractive targets for therapeutic development. Here, we report the high-resolution crystal structures of two essential glycolytic enzymes, glucose-6-phosphate isomerase (Tc PGI, 1.8 Å) and enolase (Tc enolase, 2.4 Å) and provide structural and computational analyses to support structure-based drug design. Tc PGI adopts a dimeric αβα sandwich fold and features a parasite-specific 53-residue N-terminal extension and a unique C-terminal hook region which both distinguish it from its human ortholog. Tc enolase exhibits the conserved (α/β) 8 TIM barrel fold but harbors minor distinct structural deviations, including an extended α17 helix and a structured α1 region, which differentiate it from human isoforms. Both enzymes exhibited high thermal stability, consistent with adaptation to the parasite's complex life cycle. Structure-based virtual screening using a scaffold with known multi-target potential identified distinct high-affinity inhibitors for each enzyme. Molecular dynamics simulations further confirmed stable enzyme-inhibitor interactions and favorable binding energetics. Collectively, these findings reveal structural signatures unique to T. cruzi glycolytic enzymes and lay the groundwork for the development of antiparasitic therapeutics.

Carbon-nitrogen hydrolases (CNHs) are members of the diverse nitrilase superfamily of enzymes that facilitate cellular adaptation to environmental stress by metabolizing nitrogen, detoxifying xenobiotics and catabolizing environmentally derived metabolites. Helicobacter pylori CNH (HpCNH) may contribute to metabolic flexibility under acid stress, detoxification of reactive nitrogen species or nutrient scavenging in the nutrient-limited gastric environment. Here, we report the 2.1 Å resolution crystal structure of a CNH from H. pylori strain G27 (PDB entry 6mg6). HpCNH adopts the characteristic nitrilase-superfamily αββα-sandwich core and contains the conserved catalytic cysteine typical of enzymatically active CNHs. The overall structure and active site of HpCNH are most similar to those of carbamoylputrescine amidohydrolase from the plant Medicago truncatula. Despite structural variations in loop regions, including near the active site, HpCNH retains the key residues required to bind putrescine and the prototypical N-carbamoylputrescine amidase active site.

Trypanosoma cruzi, the causative agent of Chagas disease, relies heavily on glycolysis for ATP production, making glycolytic enzymes attractive targets for therapeutic intervention. In this study, we report high-resolution crystal structures of two essential glycolytic enzymes, glucose-6-phosphate isomerase (Tc PGI; 1.8 A) and enolase (Tc enolase; 2.4 A), and integrate structural, biophysical, and computational analyses to evaluate their drug-target potential. Tc PGI adopts a dimeric alpha-beta-alpha sandwich fold and contains a parasite-specific 53-residue N-terminal extension and a distinctive C-terminal hook region that are absent in the human ortholog. Tc enolase displays the conserved (alpha/beta)8 TIM-barrel architecture but exhibits localized structural differences, including an extended alpha17 helix and a structured alpha1 region, relative to human isoforms. Both enzymes exhibit high thermal stability, consistent with adaptation to the parasite's complex life cycle. Structure-based virtual screening using a scaffold previously associated with multi-target inhibition identified candidate ligands with favorable docking scores for each enzyme. Subsequent molecular dynamics simulations and binding free-energy analyses supported stable enzyme-ligand interactions and favorable energetic profiles. Together, these results define parasite-specific structural features of two key glycolytic enzymes and provide a structural framework for future experimental validation and structure-guided development of selective antiparasitic inhibitors. ### Competing Interest Statement The authors have declared no competing interest. NIH Common Fund, 75N93022C00036

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for the discovery of new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the fifth of which is IspF. Crystal structures of Burkholderia pseudomallei IspF were determined with five different sulfonamide ligands bound. The sulfonamide-containing ligands were ethoxzolamide, acetazolamide, sulfapyridine and sulfamonomethoxine. The fifth bound ligand was a synthetic analog of acetazolamide. All ligands coordinated to the active-site Zn +2 ion through the sulfonamide group, although sulfapyridine and sulfamonomethoxine, both of which are known antibacterial agents, possess similar binding interactions that are distinct from the other three sulfonamides. These structural data will aid in the discovery of new IspF inhibitors.

The name of one of the authors in the article by Belfon et al. [(2024), Acta Cryst. F80, 348–355] is corrected.

Entamoeba histolytica causes amebiasis, a neglected disease that kills ∼100 000 people globally each year. Due to emerging drug resistance, E. histolytica is one of the target organisms for structure-based drug discovery by the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Purification, crystallization and three structures of the putative drug target endoribonuclease L-PSP from E. histolytica (EhL-PSP) are presented. EhL-PSP has a two-layer α/β-sandwich with structural homology to endoribonuclease L-PSP. All three structures reveal the prototypical YjgF/YER057c/UK114 family trimer topology with accessible allosteric active sites. Citrate molecules from the crystallization solution are bound to the allosteric site in two of the three reported structures. The large allosteric site of EhL-PSP is well conserved with bacterial YjgF/YER057c/UK114 family members and could be targeted for inhibition, drug discovery or repurposing.

Mycobacterium tuberculosis is a Gram-positive bacillus that causes tuberculosis and is a leading cause of mortality worldwide. This disease is a growing health threat due to the occurrence of multidrug resistance. Mycolic acids are essential for generating cell walls and their modification is important to the virulence and persistence of M. tuberculosis. A family of S -adenosylmethionine-dependent mycolic acid synthases modify mycolic acids and represent promising drug targets. UmaA is currently the least-understood member of this family. This paper describes the crystal structure of UmaA. UmaA is a monomer composed of two domains: a structurally conserved SAM-binding domain and a variable substrate-binding auxiliary domain. Fortuitously, our structure contains a nitrate in the active site, a structural mimic of carbonate, which is a known general base in cyclopropane-adding synthases. Further investigation indicated that the structure of the N-terminus is highly flexible. Finally, we have identified S -adenosyl- N -decyl-aminoethyl as a promising potential inhibitor.

Orotate phosphoribosyltransferase (OPRT) catalyzes the reaction that adds the pyrimidine base to the ribose in the penultimate step of the de novo biosynthesis of pyrimidine nucleotides. The OPRT structure consists of an obligate dimer, conserved throughout the phosphoribosyltransferase family. Here, we describe the structural characterization of Burkholderia cenocepacia OPRT (BcOPRT), both by X-ray crystallography and Cryo electron microscopy (Cryo-EM). While the known dimer is present in the structure of BcOPRT, a putative hexameric form was also observed by multiple methods. Analyses by chromatography, Cryo-EM, and kinetics indicate that both dimeric and hexameric forms of this enzyme are present together in solution. Comparison of the kinetics of the native protein and two variants, which were specifically designed to prevent hexamerization, reveal that only the hexameric form is enzymatically active. Collectively, these data suggest that BcOPRT may use oligomerization to control overall enzymatic activity, thus contributing to the local regulation of pyrimidine biosynthesis in this organism.

Cellular deoxyuridine 5′-triphosphate nucleotidohydrolases (dUTPases) catalyze the hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and pyrophosphate (PP i ). dUTPase is an essential metabolic enzyme which maintains the homeostatic dTTP:dUTP ratio. As DNA polymerases are unable to distinguish between thymine and uracil during replication, the dTTP:dUTP ratio is essential for preventing the misincorporation of uracil into DNA. In the absence of dUTPase regulation of the dTTP:dUTP ratio, many DNA double-strand breaks are induced by DNA-repair enzymes, which may ultimately lead to cell death. Legionnaires' disease is a rare but severe respiratory infection caused primarily by Legionella pneumophila serogroup 1. Increased characterization of the L. pneumophila proteome is of interest for the development of new treatments. Many DNA metabolism proteins have yet to be characterized in L. pneumophila , including dUTPase. Here, we present analysis of two crystal structures of L. pneumophila dUTPase in its apo and dUMP-bound states, determined to 1.80 and 1.95 Å resolution, respectively. The structures were solved by the Seattle Structural Genomics Center for Infectious Disease (SSGCID) as part of their mission to determine structures of proteins and other molecules with an important biological role in human pathogens.

Legionella pneumophila serogroup 1 is the primary causative agent of Legionnaires' disease, a rare but severe respiratory infection. While the fatality rate of Legionnaires' disease is low in the general population, it is more pronounced in vulnerable communities such as the immunocompromised. Thus, the development of new antimicrobials is of interest for use when existing antibiotics may not be applicable. Peptide deformylases (PDFs) have been under continued investigation as targets for novel antimicrobial compounds. PDF plays an essential role in protein synthesis, removing the N-terminal formyl group from new polypeptides, and is required for growth in most bacteria. Here, we report two crystal structures of L. pneumophila serogroup 1 PDF (LpPDF) bound to either Ni2+, an active state, or inhibited by actinonin and Zn2+; the structures were determined to 1.5 and 1.65 Å resolution, respectively, and were solved by the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is charged with determining structures of biologically important proteins and molecules from human pathogens. As actinonin is an antimicrobial natural product that has been used as a reference compound in drug development, these structures will help support the ongoing drug-development process.

Plasmodium vivax, a significant contributor to global malaria cases, poses an escalating health burden on a substantial portion of the world's population. The increasing spread of P. vivax because of climate change underscores the development of new and rational drug-discovery approaches. The Seattle Structural Genomics Center for Infectious Diseases is taking a structure-based approach by investigating essential enzymes such as N-myristoyltransferase (NMT). P. vivax N-myristoyltransferase (PvNMT) is a promising target for the development of novel malaria treatments unlike current drugs, which target only the erythrocytic stages of the parasite. Here, the 1.8 Å resolution ternary structure of PvNMT in complex with myristoyl-CoA and IMP-1088, a validated NMT inhibitor, is reported. IMP-1088 is a validated nonpeptidic inhibitor and a ternary complex structure with human NMT has previously been reported. IMP-1088 binds similarly to PvNMT as to human NMT.

To ensure high fidelity during protein translation, the majority of threonyl-tRNA synthetases (ThrRSs) utilize one of two types of editing domains at their N-termini to deacylate tRNAs mischarged with serine. To date, there have been few crystal structures of complete ThrRSs in the Protein Data Bank with their editing domains intact. Furthermore, there are no structures available of the complete enzyme in the apo form. Stenotrophomonas maltophilia ThrRS contains the HxxxH-type editing domain found in eukaryotes, eubacteria, and a subset of archaea. Here, we present a high-resolution crystal structure of the complete apo SmThrRS at 2.2Å resolution (PDB entry 6VU9) together with supporting small angle X-ray scattering data. The editing domain and a tRNA-binding β-hairpin in the catalytic domain are rotated away from each other in an open conformation, which is unfavorable for tRNA binding. The editing domain rotation is unique, and the structure contrasts with previously observed closed conformations showing the β-hairpin and hinge of the editing domain impinging on each other in the presence of substrates. On the basis of the available structures and sequence conservation at the engagement site, we consider a toggle clamp-like mechanism underlying the domain motions.

ABSTRACT Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO ( Nf ENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC 50 value of 0.14 ± 0.04 µM) that was toxic to trophozoites (EC 50 value of 0.21 ± 0.02 µM) while the reported CC 50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of Nf ENO with a binding affinity of –8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of Nf ENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

Brucella ovis is an etiologic agent of ovine epididymitis and brucellosis that causes global devastation in sheep, rams, goats, small ruminants and deer. There are no cost-effective methods for the worldwide eradication of ovine brucellosis. B. ovis and other protein targets from various Brucella species are currently in the pipeline for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), with the aim of identifying new therapeutic targets. Furthermore, the wealth of structures generated are effective tools for teaching scientific communication, structural science and biochemistry. One of these structures, B. ovis leucine-, isoleucine-, valine-, threonine- and alanine-binding protein (BoLBP), is a putative periplasmic amino acid-binding protein. BoLBP shares less than 29% sequence identity with any other structure in the Protein Data Bank. The production, crystallization and high-resolution structures of BoLBP are reported. BoLBP is a prototypical bacterial periplasmic amino acid-binding protein with the characteristic Venus flytrap topology of two globular domains encapsulating a large central cavity containing the peptide-binding region. The central cavity contains small molecules usurped from the crystallization milieu. The reported structures reveal the conformational flexibility of the central cavity in the absence of bound peptides. The structural similarity to other LBPs can be exploited to accelerate drug repurposing.

Griselimycin, a cyclic depsidecapeptide produced by Streptomyces griseus, is a promising lead inhibitor of the sliding clamp component of bacterial DNA polymerases (β-subunit of Escherichia coli DNA pol III). It was previously shown to inhibit the Mycobacterium tuberculosis β-clamp with remarkably high affinity and selectivity - the peptide lacks any interaction with the human sliding clamp. Here, we used a structural genomics approach to address the prospect of broader-spectrum inhibition, in particular of β-clamps from Gram-negative bacterial targets. Fifteen crystal structures of β-clamp orthologs were solved, most from Gram-negative bacteria, including eight cocrystal structures with griselimycin. The ensemble of structures samples widely diverse β-clamp architectures and reveals unique protein-ligand interactions with varying degrees of complementarity. Although griselimycin clearly co-evolved with Gram-positive β-clamps, binding affinity measurements demonstrate that the high selectivity observed previously extends to the Gram-negative orthologs, with KD values ranging from 7 to 496 nM for the wild-type orthologs considered. The collective results should aid future structure-guided development of peptide antibiotics against β-clamp proteins of a wide variety of bacterial targets.

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 μM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Nuak1 (NUAK family SnF1-like kinase-1) is a serine-threonine kinase and a member of the AMPK family. Interest in Nuak1 has increased over the years due to the role it plays in several biological processes, from tumor cell invasion and proliferation to Tau stabilization. Nuak1 is expressed in many cancer cell lines and many reports describe this target as an oncogene, the inhibition of which is hypothesized to be valuable for treating various cancer types including glioma. We report here the discovery of Nuak1 inhibitors originating from HTS hit 9 with excellent selectivity and the subsequent medicinal chemistry optimization program, supported by structural information from the first crystal structures of a Nuak1 chimeric protein which provided insights into the binding modes of our compounds. These efforts yielded a nanomolar cell potent, highly selective and brain penetrant Nuak1 inhibitor UCB9386 (56) suitable for in vivo pharmacological studies for central nervous system (CNS) disorders.

Burkholderia cenocepacia is an opportunistic human pathogen that can cause lethal infections in immunocompromised individuals, particularly those with cystic fibrosis. As such, there is a critical need to identify and characterize the structure and function of enzymes that participate in the metabolic pathways of this bacterium. Here, the high-resolution X-ray crystal structure of a short-chain dehydrogenase reductase (SDR) from B. cenocepacia J2315 (BcSDR) in complex with the coenzyme NADP+ and a benzoic acid ligand is presented. This protein has the conserved Rossmann fold of the SDR superfamily and the characteristic TGxxxGxG motif of the classical SDR subfamily. However, unlike classical SDRs, the active site of BcSDR has a leucine residue in place of the highly conserved and catalytically important tyrosine residue. Sequence analysis confirms that this leucine residue is conserved in this SDR across the Burkholderiales order. This suggests that BcSDR is more appropriately classified into the divergent SDR subfamily. In addition, this enzyme would necessarily employ a different enzyme mechanism to that proposed as a general mechanism for most SDRs.