Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections.

Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in Staphylococcus aureus Support a Role in Bacterial Stress Response.

The development of new treatment options for bacterial infections requires access to new targets for antibiotics and antivirulence strategies. Chemoproteomic approaches are powerful tools for profiling and identifying novel druggable target candidates, but their functions often remain uncharacterized. Previously, we used activity-based protein profiling in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred to as yvaK in Bacillus subtilis) at the molecular and cellular level. First, phenotypic characterization of fphH-deficient transposon mutants revealed phenotypes during growth under nutrient deprivation, biofilm formation, and intracellular survival. Biochemical and structural investigations revealed that FphH acts as an esterase and lipase based on a fold well suited to act on a small to long hydrophobic unbranched lipid group within its substrate and can be inhibited by active site-targeting oxadiazoles. Prompted by a previous observation that fphH expression was upregulated in response to fusidic acid, we found that FphH can deacetylate this ribosome-targeting antibiotic, but the lack of FphH function did not infer major changes in antibiotic susceptibility. In conclusion, our results indicate a functional role of this hydrolase in S. aureus stress responses, and hypothetical functions connecting FphH with components of the ribosome rescue system that are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate the development of specific FphH inhibitors and probes to elucidate its physiological role and validity as a drug target.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections.

Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolonebased activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

Newly discovered Staphylococcus aureus serine hydrolase probe and drug targets.

There is an urgent need for new diagnosis and treatment options for the bacterial pathogen Staphylococcus aureus. This review will summarize data on ten recently discovered biofilm-associated serine hydrolases called fluorophosphonate-binding hydrolases (FphA-J). Based on the summarized findings, many of these proteins represent intriguing new targets for probe and drug development.

Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome.

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity.

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

Identification of highly selective covalent inhibitors by phage display.

Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic 'warhead' to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.

Uncovering a superfamily of nickel-dependent hydroxyacid racemases and epimerases.

Isomerization reactions are fundamental in biology. Lactate racemase, which isomerizes L- and D-lactate, is composed of the LarA protein and a nickel-containing cofactor, the nickel-pincer nucleotide (NPN). In this study, we show that LarA is part of a superfamily containing many different enzymes. We overexpressed and purified 13 lactate racemase homologs, incorporated the NPN cofactor, and assayed the isomerization of different substrates guided by gene context analysis. We discovered two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxyacid racemase among the tested enzymes. We solved the structure of a malate racemase apoprotein and used it, along with the previously described structures of lactate racemase holoprotein and D-gluconate epimerase apoprotein, to identify key residues involved in substrate binding. This study demonstrates that the NPN cofactor is used by a diverse superfamily of α-hydroxyacid racemases and epimerases, widely expanding the scope of NPN-dependent enzymes.

Molecular Regulation of the Polycomb Repressive-Deubiquitinase.

Post-translational modification of histone proteins plays a major role in histone-DNA packaging and ultimately gene expression. Attachment of ubiquitin to the C-terminal tail of histone H2A (H2AK119Ub in mammals) is particularly relevant to the repression of gene transcription, and is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here, we outline recent advances in the understanding of PR-DUB regulation, which have come through structural studies of the Drosophila melanogaster PR-DUB, biochemical investigation of the human PR-DUB, and functional studies of proteins that associate with the PR-DUB. In humans, mutations in components of the PR-DUB frequently give rise to malignant mesothelioma, melanomas, and renal cell carcinoma, and increase disease risk from carcinogens. Diverse mechanisms may underlie disruption of the PR-DUB across this spectrum of disease. Comparing and contrasting the PR-DUB in mammals and Drosophila reiterates the importance of H2AK119Ub through evolution, provides clues as to how the PR-DUB is dysregulated in disease, and may enable new treatment approaches in cancers where the PR-DUB is disrupted.

Structural Basis for the Inhibitor and Substrate Specificity of the Unique Fph Serine Hydrolases of Staphylococcus aureus.

Staphylococcus aureus is a prevalent bacterial pathogen in both community and hospital settings, and its treatment is made particularly difficult by resilience within biofilms. Within this niche, serine hydrolase enzymes play a key role in generating and maintaining the biofilm matrix. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fph's), some of which contribute to the virulence of S. aureus in vivo. These 10 Fph proteins have limited annotation and have few, if any, characterized bacterial or mammalian homologues. This suggests unique hydrolase functions even within bacterial species. Here we report structures of one of the most abundant Fph family members, FphF. Our structures capture FphF alone, covalently bound to a substrate analogue and bound to small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. In line with these findings, we show that FphF has promiscuous esterase activity toward hydrophobic lipid substrates. We present docking studies that characterize interactions of inhibitors and substrates within the active site environment, which can be extended to other Fph family members. Comparison of FphF to other esterases and the wider Fph protein family suggest that FphF forms a new esterase subfamily. Our data suggest that other Fph enzymes, including the virulence factor FphB, are likely to have more restricted substrate profiles than FphF. This work demonstrates a clear molecular rationale for the specificity of fluorophosphonate probes that target FphF and provides a structural template for the design of enhanced probes and inhibitors of the Fph family of serine hydrolases.

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function.

The lanthanide elements (Ln3+), those with atomic numbers 57-63 (excluding promethium, Pm3+), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what features distinguish enzymes that use Ln3+-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La3+ bound in the active-site region and coordinated by Asp320 Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp320 is needed for in vivo catalytic function, in vitro activity, and La3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca2+ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La3+ These results provide evidence that a Ln3+-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

Crystallographic characterization of a tri-Asp metal-binding site at the three-fold symmetry axis of LarE.

Detailed crystallographic characterization of a tri-aspartate metal-binding site previously identified on the three-fold symmetry axis of a hexameric enzyme, LarE from Lactobacillus plantarum, was conducted. By screening an array of monovalent, divalent, and trivalent metal ions, we demonstrated that this metal binding site stoichiometrically binds Ca2+, Mn2+, Fe2+/Fe3+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+, but not monovalent metal ions, Cr3+, Mg2+, Y3+, Sr2+ or Ba2+. Extensive database searches resulted in only 13 similar metal binding sites in other proteins, indicative of the rareness of tri-aspartate architectures, which allows for engineering such a selective multivalent metal ion binding site into target macromolecules for structural and biophysical characterization.

A structural perspective on the PP-loop ATP pyrophosphatase family.

Derived from an ancient ATP-hydrolyzing Rossmann-like fold protein, members of the PP-loop ATP pyrophosphatase family feature an absolutely conserved P-loop-like "SxGxDS/T" motif used for binding and presenting ATP for substrate adenylylation (AMPylation). Since the first family member was reported more than 20 years ago, numerous representatives catalyzing very diverse reactions have been characterized both functionally and structurally. The availability of more than 100 high quality structures in the protein data bank provides an excellent opportunity to gain structural insights into the generally conserved catalytic mechanism and the uniqueness of the reactions catalyzed by family members. In this work, we conducted a thorough database search for the PP-loop ATP pyrophosphatase family members, resulting in the most comprehensive and up-to-date collection that includes 18 enzyme families. Structure comparison of representative family members allowed us to identify common structure features in the core catalytic domain, as well as four highly variable regions that define the unique chemistry for each enzyme family. The newly identified enzymes, particularly those from pathogens, warrant further research to enlarge the scope of this ever-expanding and highly diverse enzyme superfamily for use in potential bioengineering and biomedical applications.

Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum.

LarE from Lactobacillus plantarum is an ATP-dependent sulfur transferase that sacrifices its Cys176 sulfur atom to form a dehydroalanine (Dha) side chain during biosynthesis of the covalently linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide) of lactate racemase. Coenzyme A (CoA) stabilizes LarE and forms a CoA-Cys176 mixed disulfide with the protein. This study presents the crystal structure of the LarE/CoA complex, revealing protein interactions with CoA that mimic those for binding ATP. CoA weakly inhibits LarE activity, and the persulfide of CoA is capable of partially regenerating functional LarE from the Dha176 form of the protein. The physiological relevance of this cycling reaction is unclear. A new form of LarE was discovered, an NPN-LarE covalent adduct, explaining prior results in which activation of the lactate racemase apoprotein required only the isolated LarE. The crystal structure of the inactive C176A variant revealed a fold essentially identical to that of wild-type LarE. Additional active site variants of LarE were created and their activities characterized, with all LarE variants analyzed in terms of the structure. Finally, the L. plantarum LarE structure was compared to a homology model of Thermoanaerobacterium thermosaccharolyticum LarE, predicted to contain three cysteine residues at the active site, and to other proteins with a similar fold and multiple active site cysteine residues. These findings suggest that some LarE orthologs may not be sacrificial but instead may catalyze sulfur transfer by using a persulfide mechanism or from a labile site on a [4Fe-4S] cluster at this position.

Nickel-pincer nucleotide cofactor.

A novel organometallic cofactor, nickel pyridinium-3,5-dithiocarboxylic acid mononucleotide, was recently discovered in lactate racemase (LarA) of Lactobacillus plantarum. This review summarizes the substantial progress made in uncovering the function of this cofactor as a transient hydride acceptor in the LarA mechanism. The latest developments related to cofactor biosynthesis reveal insights into a pathway in which LarB serves as a nicotinic acid adenine dinucleotide hydrolase/carboxylase, LarE acts as a sacrificial sulfur transferase, and LarC functions as a nickel insertase, forming the nickel-pincer nucleotide cofactor that becomes covalently tethered to LarA in some bacteria. Bioinformatic studies reveal a widespread occurrence of larA, larB, larC, and larE orthologs in microorganisms, and additional roles for the cofactor are considered.

Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase.

Bacterial lactate racemase is a nickel-dependent enzyme that contains a cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN). The LarC enzyme from the bacterium Lactobacillus plantarum participates in NPN biosynthesis by inserting nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide. This reaction, known in organometallic chemistry as a cyclometalation, is characterized by the formation of new metal-carbon and metal-sulfur σ bonds. LarC is therefore the first cyclometallase identified in nature, but the molecular mechanism of LarC-catalyzed cyclometalation is unknown. Here, we show that LarC activity requires Mn2+-dependent CTP hydrolysis. The crystal structure of the C-terminal domain of LarC at 1.85 Å resolution revealed a hexameric ferredoxin-like fold and an unprecedented CTP-binding pocket. The loss-of-function of LarC variants with alanine variants of acidic residues leads us to propose a carboxylate-assisted mechanism for nickel insertion. This work also demonstrates the in vitro synthesis and purification of the NPN cofactor, opening new opportunities for the study of this intriguing cofactor and of NPN-utilizing enzymes.

Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism.

Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( kH/ kD of 3.11 ± 0.17) was established using 2-α-2H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.

Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway.

Zrt/Irt-like proteins (ZIPs) play fundamental roles in metal metabolism/homeostasis and are broadly involved in numerous physiological and pathological processes. The lack of high-resolution structure of the ZIPs hinders understanding of the metal transport mechanism. We report two crystal structures of a prokaryotic ZIP in lipidic cubic phase with bound metal substrates (Cd2+ at 2.7 Å and Zn2+ at 2.4 Å). The structures revealed a novel 3+2+3TM architecture and an inward-open conformation occluded at the extracellular side. Two metal ions were trapped halfway through the membrane, unexpectedly forming a binuclear metal center. The Zn2+-substituted structure suggested asymmetric functions of the two metal-binding sites and also revealed a route for zinc release. Mapping of disease-causing mutations, structure-guided mutagenesis, and cell-based zinc transport assay demonstrated the crucial role of the binuclear metal center for human ZIP4. A metal transport mechanism for the ZIP from Bordetella bronchiseptica was proposed, which is likely applicable to other ZIPs.

Structural insights into the catalytic mechanism of a sacrificial sulfur insertase of the N-type ATP pyrophosphatase family, LarE.

The lar operon in Lactobacillus plantarum encodes five Lar proteins (LarA/B/C/D/E) that collaboratively synthesize and incorporate a niacin-derived Ni-containing cofactor into LarA, an Ni-dependent lactate racemase. Previous studies have established that two molecules of LarE catalyze successive thiolation reactions by donating the sulfur atom of their exclusive cysteine residues to the substrate. However, the catalytic mechanism of this very unusual sulfur-sacrificing reaction remains elusive. In this work, we present the crystal structures of LarE in ligand-free and several ligand-bound forms, demonstrating that LarE is a member of the N-type ATP pyrophosphatase (PPase) family with a conserved N-terminal ATP PPase domain and a unique C-terminal domain harboring the putative catalytic site. Structural analysis, combined with structure-guided mutagenesis, leads us to propose a catalytic mechanism that establishes LarE as a paradigm for sulfur transfer through sacrificing its catalytic cysteine residue.