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Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express Na -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N5, N5-di-methyl Na-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 χ 104M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 μM; Ki = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4(diethylamino)phenylboronic acid (IC50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn). [ABSTRACT FROM AUTHOR]

The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity (1,2) and potential role in human disease (3). These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic (4). The carboxyterminal region of the C protein is the main cytotoxic component (5), and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells (5). Toxin complexes have been visualized using single-particle electron microscopy (6,7), but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a β-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats (8), and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins (9). The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery., ABC toxins were first identified, and have been best characterized, in the bacterium Photorhabdus luminescens (1). However, the entomopathogenic bacterium Yersinia entomophaga contains a related toxin complex locus that includes [...]

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 Å resolution. The structure revealed an elongated β‐barrel fold enclosing two distinct cavities and one α‐helix extending away from the β‐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 β‐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. [ABSTRACT FROM AUTHOR]

The temperate coliphage [lambda] after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis/lysogeny decision is the homotetrameric transcription-activator protein CII (4 x 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-[N.sub.6]-T-T-G-C at each of the three phage promoters it activates: [p.sub.E], [p.sub.I], and [p.sub.aQ]. Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-[Angstrom] resolution. The CII monomer is comprised of four [lambda] helices and a disordered C terminus. The first three helices ([alpha]1-[alpha]3) form a compact domain, whereas the fourth helix ([alpha]4) protrudes in different orientations in each subunit. A four-helix bundle, formed by [alpha]4 from each subunit, holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the inverted-repeat sequences of most prokaryotic activators. direct-repeat recognition | helix-turn-helix motif | transcription activator

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. [ABSTRACT FROM AUTHOR]

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 R1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn–Teller-distorted Cu octa­hedra. The sulfate tetrahedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetra­hedra are split and rotated, which enables additional hydrogen bonds. [ABSTRACT FROM AUTHOR]

The rise of antibiotic resistance combined with the lack of new products entering the market has led to bacterial infections becoming one of the biggest threats to global health. Therefore, there is an urgent need to identify novel antibiotic targets, such as dihydrodipicolinate synthase (DHDPS), an enzyme involved in the production of essential metabolites in cell wall and protein synthesis. Here, we utilised a 7‐residue sequence motif to identify mis‐annotation of multiple DHDPS genes in the high‐priority Gram‐negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. We subsequently confirmed these mis‐annotations using a combination of enzyme kinetics and X‐ray crystallography. Thus, this study highlights the need to ensure genes encoding promising drug targets, like DHDPS, are annotated correctly, especially for clinically important pathogens. PDB ID: 6UE0. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. Databases: PDB ID: 6P90. [ABSTRACT FROM AUTHOR]

The specific roles for acid-base catalysis is strictosidine synthase are described by using the data derived from kinetic isotope effects (KIE), rate dependence of pH, and comparisons to a nonenzymatic Pictet-Spengler reaction. The studies have shown that a spiroindolenine intermediate, which is often invoked in the Pictet-Spengler mechanism, does not occur.

Two commonly encountered bottlenecks in the structure determination of a protein by X‐ray crystallography are screening for conditions that give high‐quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5‐amino‐2,4,6‐triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high‐quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg‐white lysozyme and the N‐terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion‐associated lysin or endolysin. [ABSTRACT FROM AUTHOR]

MX2 is an in‐vacuum undulator‐based crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X‐rays in the energy range 4.8–21 keV to a focal spot of 22 × 12 µm FWHM (H × V). At 13 keV the flux at the sample is 3.4 × 1012 photons s−1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard‐to‐crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights. [ABSTRACT FROM AUTHOR]

The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a diversity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the diversity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni ( CjeATP-PRT). This enzyme is a member of the long form (HisGL) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. [ABSTRACT FROM AUTHOR]