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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 Nα-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 Nα-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 x 104 M-1 s-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; K[sub i] = 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