On the function of TRAP substrate-binding proteins: conformational variation of the sialic acid binding protein SiaP.

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding protein to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 µM) compared to N-glycolylneuraminate (KD = 4.4 µM), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, we suggest that the open unliganded conformation may sample multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

Engineering broad-spectrum inhibitors of inflammatory chemokines from subclass A3 tick evasins.

Chemokines are key regulators of leukocyte trafficking and attractive targets for anti-inflammatory therapy. Evasins are chemokine-binding proteins from tick saliva, whose application as anti-inflammatory therapeutics will require manipulation of their chemokine target selectivity. Here we describe subclass A3 evasins, which are unique to the tick genus Amblyomma and distinguished from "classical" class A1 evasins by an additional disulfide bond near the chemokine recognition interface. The A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. Unlike A1 evasins, EVA-A was not highly dependent on N- and C-terminal regions to differentiate chemokine targets. Structures of chemokine-bound EVA-A revealed a deep hydrophobic pocket, unique to A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine. Mutations to this pocket altered the chemokine selectivity of EVA-A. Thus, class A3 evasins provide a suitable platform for engineering proteins with applications in research, diagnosis or anti-inflammatory therapy.

Characterization of a novel recombinant calcium-binding protein from Arca subcrenata and its anti-hepatoma activities in vitro and in vivo.

Previous studies demonstrated that ASP-3 was a novel calcium-binding protein from Arca subcrenata that effectively inhibited the proliferation of HepG2 cells. To further study the antitumor activity and mechanism of ASP-3, the cytotoxic effects of recombinant ASP-3 were evaluated in HepG2 cells. The results demonstrated that ASP-3 inhibited the proliferation of HepG2 cells by competitively binding to the EGF binding pocket of EGFR and inhibiting the JAK-STAT, RAS-RAF-MEK-ERK, and PI3K-Akt-mTOR signaling pathways mediated by EGFR. ASP-3 significantly inhibited tumor growth in a HepG2 cell subcutaneous xenograft nude mouse model, and its (25 mg/kg and 75 mg/kg) tumor inhibition rates were 46.92 % and 60.28 %, respectively. Furthermore, the crystal structure of ASP-3 was resolved at 1.4 Å. ASP-3 formed as a stable dimer and folded as an EF-Hand structure. ASP-3 stably bound to domain I and domain III of the EGFR extracellular region by using molecular docking and molecular dynamics simulation analysis. Compared with the endogenous ligand EGF, ASP-3 displayed a stronger interaction with EGFR. These experimental results indicated that recombinant ASP-3 possessed an effective anti-hepatoma effect. So, it might be a potential molecule for liver cancer therapy.

Repurposed inhibitor of bacterial dihydrodipicolinate reductase exhibits effective herbicidal activity.

Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicide-resistant weeds. Here, we employed a novel strategy to repurpose a 'failed' antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana. We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana. We also showed that our lead compound is the first lysine biosynthesis inhibitor with activity against both monocotyledonous and dicotyledonous weed species, by demonstrating its effectiveness at reducing the germination and growth of Lolium rigidum (rigid ryegrass) and Raphanus raphanistrum (wild radish). These results provide proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing 'failed' antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes.

Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function.

Autotransporters (ATs) are a large family of bacterial secreted and outer membrane proteins that encompass a wide range of enzymatic activities frequently associated with pathogenic phenotypes. We present the structural and functional characterisation of a subtilase autotransporter, Ssp, from the opportunistic pathogen Serratia marcescens. Although the structures of subtilases have been well documented, this subtilisin-like protein is associated with a 248 residue ß-helix and itself includes three finger-like protrusions around its active site involved in substrate interactions. We further reveal that the activity of the subtilase AT is required for entry into epithelial cells as well as causing cellular toxicity. The Ssp structure not only provides details about the subtilase ATs, but also reveals a common framework and function to more distantly related ATs. As such these findings also represent a significant step forward toward understanding the molecular mechanisms underlying the functional divergence in the large AT superfamily.

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families.

CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Crystal structure of the putative cell-wall lipoglycan biosynthesis protein LmcA from Mycobacterium smegmatis.

The bacterial genus Mycobacterium includes important pathogens, most notably M. tuberculosis, which infects one-quarter of the entire human population, resulting in around 1.4 million deaths from tuberculosis each year. Mycobacteria, and the closely related corynebacteria, synthesize a class of abundant glycolipids, the phosphatidyl-myo-inositol mannosides (PIMs). PIMs serve as membrane anchors for hyperglycosylated species, lipomannan (LM) and lipoarabinomannan (LAM), which are surface-exposed and modulate the host immune response. Previously, in studies using the model species Corynebacterium glutamicum, NCgl2760 was identified as a novel membrane protein that is required for the synthesis of full-length LM and LAM. Here, the first crystal structure of its ortholog in Mycobacterium smegmatis, MSMEG_0317, is reported at 1.8 Šresolution. The structure revealed an elongated ß-barrel fold enclosing two distinct cavities and one α-helix extending away from the ß-barrel core, resembling a `cone with a flake' arrangement. Through xenon derivatization and structural comparison with AlphaFold2-derived predictions of the M. tuberculosis homolog Rv0227c, structural elements were identified that may undergo conformational changes to switch from `closed' to `open' conformations, allowing cavity access. An AlphaFold2-derived NCgl2760 model predicted a smaller ß-barrel core with an enclosed central cavity, suggesting that all three proteins, which were collectively termed LmcA, may have a common mechanism of ligand binding through these cavities. These findings provide new structural insights into the biosynthetic pathway for a family of surface lipoglycans with important roles in mycobacterial pathogenesis.

ß-Methyltryptamine Provoking the Crucial Role of Strictosidine Synthase Tyr151-OH for Its Stereoselective Pictet-Spengler Reactions to Tryptoline-type Alkaloids.

Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).

Graphene and Graphene Oxide as a Support for Biomolecules in the Development of Biosensors.

Graphene and graphene oxide have become the base of many advanced biosensors due to their exceptional characteristics. However, lack of some properties, such as inertness of graphene in organic solutions and non-electrical conductivity of graphene oxide, are their drawbacks in sensing applications. To compensate for these shortcomings, various methods of modifications have been developed to provide the appropriate properties required for biosensing. Efficient modification of graphene and graphene oxide facilitates the interaction of biomolecules with their surface, and the ultimate bioconjugate can be employed as the main sensing part of the biosensors. Graphene nanomaterials as transducers increase the signal response in various sensing applications. Their large surface area and perfect biocompatibility with lots of biomolecules provide the prerequisite of a stable biosensor, which is the immobilization of bioreceptor on transducer. Biosensor development has paramount importance in the field of environmental monitoring, security, defense, food safety standards, clinical sector, marine sector, biomedicine, and drug discovery. Biosensor applications are also prevalent in the plant biology sector to find the missing links required in the metabolic process. In this review, the importance of oxygen functional groups in functionalizing the graphene and graphene oxide and different types of functionalization will be explained. Moreover, immobilization of biomolecules (such as protein, peptide, DNA, aptamer) on graphene and graphene oxide and at the end, the application of these biomaterials in biosensors with different transducing mechanisms will be discussed.

Towards novel herbicide modes of action by inhibiting lysine biosynthesis in plants.

Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides with novel modes of action are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. The first class of plant DHDPS inhibitors with micromolar potency against Arabidopsis thaliana DHDPS was identified using a high-throughput chemical screen. We determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. The inhibitors also attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide-resistant weeds.

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism.

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

Helicobacter pylori Xanthine-Guanine-Hypoxanthine Phosphoribosyltransferase-A Putative Target for Drug Discovery against Gastrointestinal Tract Infections.

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.

Differential lysine-mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms.

Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate-limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l-lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing > 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine-mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10-fold more sensitive to lysine. We subsequently used X-ray crystallography to probe for structural differences between the apo- and lysine-bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6-fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. DATABASES: Structures described are available in the Protein Data Bank under the accession numbers 6VVH and 6VVI.

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity.

Plantacyclin B21AG is a circular bacteriocin produced by Lactiplantibacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from a food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 18 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed that Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. The Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the relative importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.

Structural basis for differentiation between two classes of thiolase: Degradative vs biosynthetic thiolase.

Thiolases are a well characterized family of enzymes with two distinct categories: degradative, ß-ketoadipyl-CoA thiolases and biosynthetic, acetoacetyl-CoA thiolases. Both classes share an identical catalytic triad but catalyze reactions in opposite directions. Moreover, it is established that in contrast to the biosynthetic thiolases the degradative thiolases can accept substrates with broad chain lengths. Hitherto, no residue or structural pattern has been recognized that might help to discern the two thiolases, here we exploit, a tetrameric degradative thiolase from Pseudomonas putida KT2440 annotated as PcaF, as a model system to understand features which distinguishes the two classes using structural studies and bioinformatics analyses. Degradative thiolases have different active site architecture when compared to biosynthetic thiolases, demonstrating the dissimilar chemical nature of the active site architecture. Both thiolases deploy different "anchoring residues" to tether the large Coenzyme A (CoA) or CoA derivatives. Interestingly, the H356 of the catalytic triad in PcaF is directly involved in tethering the CoA/CoA derivatives into the active site and we were able to trap a gridlocked thiolase structure of the H356A mutant, where the CoA was found to be covalently linked to the catalytic cysteine residue, inhibiting the overall reaction. Further, X-ray structures with two long chain CoA derivatives, hexanal-CoA and octanal-CoA helped in delineating the long tunnel of 235 Å2 surface area in PcaF and led to identification of a unique covering loop exclusive to degradative thiolases that plays an active role in determining the tunnel length and the nature of the binding substrate.

Crystal structure of posnjakite formed in the first crystal water-cooling line of the ANSTO Melbourne Australian Synchrotron MX1 Double Crystal Monochromator.

Exceptionally large crystals of posnjakite, Cu4SO4(OH)6(H2O), formed during corrosion of a Swagelock(tm) Snubber copper gasket within the MX1 beamline at the ANSTO-Melbourne, Australian Synchrotron. The crystal structure was solved using synchrotron radiation to R 1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn-Teller-distorted Cu octa-hedra. The sulfate tetra-hedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetra-hedra are split and rotated, which enables additional hydrogen bonds.

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.