Activation of zinc uptake regulator by zinc binding to three regulatory sites.

Zur is a Fur-family metalloregulator that is widely used to control zinc homeostasis in bacteria. In Streptomyces coelicolor, Zur (ScZur) acts as both a repressor for zinc uptake (znuA) gene and an activator for zinc exporter (zitB) gene. Previous structural studies revealed three zinc ions specifically bound per ScZur monomer; a structural one to allow dimeric architecture and two regulatory ones for DNA-binding activity. In this study, we present evidence that Zur contains a fourth specific zinc-binding site with a key histidine residue (H36), widely conserved among actinobacteria, for regulatory function. Biochemical, genetic, and calorimetric data revealed that H36 is critical for hexameric binding of Zur to the zitB zurbox and further binding to its upstream region required for full activation. A comprehensive thermodynamic model demonstrated that the DNA-binding affinity of Zur to both znuA and zitB zurboxes is remarkably enhanced upon saturation of all three regulatory zinc sites. The model also predicts that the strong coupling between zinc binding and DNA binding equilibria of Zur drives a biphasic activation of the zitB gene in response to a wide concentration change of zinc. Similar mechanisms may be pertinent to other metalloproteins, expanding their response spectrum through binding multiple regulatory metals.

An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Publisher Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Analyses of Cetacean Virus-Responsive Genes Reveal Evolutionary Marks in Mucosal Immunity-Associated Genes.

Viruses are the most common and abundant organisms in the marine environment. To better understand how cetaceans have adapted to this virus-rich environment, we compared cetacean virus-responsive genes to those from terrestrial mammals. We identified virus-responsive gene sequences in seven species of cetaceans, which we compared with orthologous sequences in seven terrestrial mammals. As a result of evolution analysis using the branch model and the branch-site model, 21 genes were selected using at least one model. IFN-ε, an antiviral cytokine expressed at mucous membranes, and its receptor IFNAR1 contain cetacean-specific amino acid substitutions that might change the interaction between the two proteins and lead to regulation of the immune system against viruses. Cetacean-specific amino acid substitutions in IL-6, IL-27, and the signal transducer and activator of transcription (STAT)1 are also predicted to alter the mucosal immune response of cetaceans. Since mucosal membranes are the first line of defense against the external environment and are involved in immune tolerance, our analysis of cetacean virus-responsive genes suggests that genes with cetacean-specific mutations in mucosal immunity-related genes play an important role in the protection and/or regulation of immune responses against viruses.

Author Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

In the version of this article originally published, reference to another structure of GenB1 was omitted (Dow, G. T., Thoden, J. B., & Holden, H. M. The three-dimensional structure of NeoB: an aminotransferase involved in the biosynthesis of neomycin. Protein Sci. 27, 945-956 (2018)). This paper is now cited as reference 32, and "Another structure of GenB1 was also reported independently during the revision of this article32" was added to the text in the Discussion section. This error has been corrected in the PDF and HTML versions of the article.

Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora. Though the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora. The narrow substrate specificity of a key glycosyltransferase and the C6'-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2'-deamination enzymes, limits GB production in M. echinospora. The crystal structure of the aminotransferase involved in C6'-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

Antibacterial Meroterpenoids, Merochlorins G-J from the Marine Bacterium Streptomyces sp.

Four new chlorinated meroterpenoids, merochlorins G-J (1-4), and 10, a dihydronaphthalenedione precursor, along with known merochlorins A (5) and C-F (6-9), were obtained from cultivation of the bacterium strain Streptomyces sp. CNH-189, which was isolated from marine sediment. The planar structures of compounds 1-4 and 10 were elucidated by interpretation of MS, UV, and NMR spectroscopic data. The relative configurations of compounds 1-4 were determined via analysis of nuclear Overhauser effect (NOE) spectroscopic data, after which their absolute configurations were established by comparing the experimental electronic circular dichroism (ECD) spectra of compounds 1-4 to those of previously reported possible enantiomer models and DP4 calculations. Compound 3 displayed strong antibacterial activities against Bacillus subtilis, Kocuria rhizophila, and Staphylococcus aureus, with MIC values of 1, 2, and 2 µg/mL, respectively, whereas compound 1 exhibited weak antibacterial effects on these three strains, with a 16-32 µg/mL MIC value range.

Saccharobisindole, Neoasterric Methyl Ester, and 7-Chloro-4(1H)-quinolone: Three New Compounds Isolated from the Marine Bacterium Saccharomonospora sp.

Analysis of the chemical components from the culture broth of the marine bacterium Saccharomonospora sp. CNQ-490 has yielded three novel compounds: saccharobisindole (1), neoasterric methyl ester (2), and 7-chloro-4(1H)-quinolone (3), in addition to acremonidine E (4), pinselin (5), penicitrinon A (6), and penicitrinon E (7). The chemical structures of the three novel compounds were elucidated by the interpretation of 1D, 2D nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) data. Compound 2 generated weak inhibition activity against Bacillus subtilis KCTC2441 and Staphylococcus aureus KCTC1927 at concentrations of 32 µg/mL and 64 µg/mL, respectively, whereas compounds 1 and 3 did not have any observable effects. In addition, compound 2 displayed weak anti-quorum sensing (QS) effects against S. aureus KCTC1927 and Micrococcus luteus SCO560.

DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition.

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

Unprecedented Noncanonical Features of the Nonlinear Nonribosomal Peptide Synthetase Assembly Line for WS9326A Biosynthesis.

Systematic inactivation of nonribosomal peptide synthetase (NRPS) domains and translocation of the thioesterase (TE) domain revealed several unprecedented nonlinear NRPS assembly processes during the biosynthesis of the cyclodepsipeptide WS9326A in Streptomyces sp. SNM55. First, two sets of type ΙΙ TE (TEΙΙ)-like enzymes mediate the shuttling of activated amino acids between two sets of stand-alone adenylation (A)-thiolation (T) didomain modules and an "A-less" condensation (C)-T module with distinctive specificities and flexibilities. This was confirmed by the elucidation of the affinities of the A-T didomains for the TEΙΙs and its structure. Second, the C-T didomain module operates iteratively and independently from other modules in the same protein to catalyze two chain elongation cycles. Third, this biosynthetic pathway includes the first example of module skipping, where the interpolated C and T domains are required for chain transfer.

Novel inhibition mechanism of carbapenems on the ACC-1 class C ß-lactamase.

The hydrolysis of ß-lactam antibiotics by class C ß-lactamases proceeds through the acylation and the rate-determining deacylation steps mediated by the nucleophilic serine and the deacylation water, respectively. The pose of poor substrates such as carbapenems in the acylated enzyme is responsible for the low efficient deacylation reaction. Here we present the crystal structures of the Y150F variant of the ACC-1 class C ß-lactamase in the apo and acylated states. In the acylated enzyme complexed with two carbapenems, imipenem and meropenem, the lactam carbonyl oxygen is located in the oxyanion hole. However, the five-membered pyrroline ring displays a novel orientation that has not been reported so far. The ring is rotated such that its C3 carboxylate makes salt bridges with Lys67 and Ly315, which is accompanied by the side-chain rotamer change of Phe150. The C3 carboxylate is placed where the deacylation water occupies in the apo-enzyme, which, together with the displacement of the catalytic base residue at position 150, explains why carbapenems are poor substrates of ACC-1.

Structural Insights into Catalytic Relevances of Substrate Poses in ACC-1.

ACC-1 is a plasmid-encoded class C ß-lactamase identified in clinical isolates of Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, and Escherichia coli ACC-1-producing bacteria are susceptible to cefoxitin, whereas they are resistant to oxyimino cephalosporins. Here, we depict crystal structures of apo ACC-1, adenylylated ACC-1, and acylated ACC-1 complexed with cefotaxime and cefoxitin. ACC-1 has noteworthy structural alterations in the R2 loop, the Ω loop, and the Phe119 loop located along the active-site rim. The adenylate covalently bonded to the nucleophilic serine reveals a tetrahedral phosphorus mimicking the deacylation transition state. Cefotaxime in ACC-1 has a proper conformation for the substrate-assisted catalysis in that its C-4 carboxylate and N-5 nitrogen are adequately located to facilitate the deacylation reaction. In contrast, cefoxitin in ACC-1 has a distinct conformation, in which those functional groups cannot contribute to catalysis. Furthermore, the orientation of the deacylating water relative to the acyl carbonyl group in ACC-1 is unfavorable for nucleophilic attack.

Structural basis for the interaction between DJ-1 and Bcl-XL.

DJ-1 is a multifunctional protein associated with Parkinson's disease (PD) and tumorigenesis. In response to ultraviolet B (UVB) irradiation, DJ-1 is translocated into the mitochondria, and its interaction with the mitochondrial protein Bcl-XL protects cells against death. In this study, we characterized the molecular interaction between DJ-1 and Bcl-XL by NMR spectroscopy. The NMR chemical shift perturbation data demonstrated that the oxidized but not the reduced form of DJ-1 binds to the predominantly hydrophobic groove surrounded by the BH1-BH3 domains in Bcl-XL. In addition, our results showed that the C-terminal α8-helix peptide (Cpep) of DJ-1 binds to the pro-apoptotic BH3 peptide-binding hydrophobic groove in Bcl-XL and, thus, acts as a Bcl-XL-binding motif. In combination with the NMR chemical shift perturbation data, a refined structural model of the Bcl-XL/DJ-1 Cpep complex revealed that the binding mode is remarkably similar to that of other Bcl-XL/pro-apoptotic BH3 peptide complexes. Taken together, our results provide a structural basis for the binding mechanism between DJ-1 and Bcl-XL, which will contribute to molecular understanding of the role of mitochondrial DJ-1 in Bcl-XL regulation in response to oxidative stress.

GMP and IMP Are Competitive Inhibitors of CMY-10, an Extended-Spectrum Class C ß-Lactamase.

Nucleotides were effective in inhibiting the class C ß-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a Ki value of 16.2 µM. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5'-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.

Structural and mechanistic insights into the inhibition of class C ß-lactamases through the adenylylation of the nucleophilic serine.

Objectives: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C ß-lactamases. Methods: : The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates. Results: : The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1 s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria. Conclusions: : Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C ß-lactamase inhibitors on the basis of the nucleotide scaffold.

Crystal structure of EstSRT1, a family VIII carboxylesterase displaying hydrolytic activity toward oxyimino cephalosporins.

EstSRT1 is a family VIII carboxylesterase that hydrolyzes oxyimino third- and fourth-generation cephalosporins, first-generation cephalosporins and ester substrates. According to the crystal structure of EstSRT1 (2.0-Å resolution), this protein contains a large α/ß domain and a small α-helical domain and harbors three catalytic residues (Ser71, Lys74, and Tyr160) in the cavity at the domain interface, similarly to other family VIII carboxylesterases. Comparison of the structures of EstSRT1 and EstU1, a family VIII carboxylesterase with no hydrolytic activity toward bulky oxyimino cephalosporins, revealed that EstSRT1 has a smaller active site, despite its extended substrate range. The B-factors of the active site segments that could potentially contact with the oxyimino groups and the R2 side chains of oxyimino cephalosporins are higher in EstSRT1 than in EstU1, thus suggesting the role of the active site's structural flexibility in the extension of EstSRT1's substrate spectrum.

Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α.

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568-596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574-589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.

Cross-protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv.

Gram-negative Vibrio species secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross-reactivity and cross-protectivity of mAbs against V. vulnificus RtxA1/MARTXVv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTXVc , inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.

Formate-driven growth coupled with H(2) production.

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H(2) (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol(-1)) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H(2) consumption by the methanogens, which lowers the H(2) partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H(2). Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H(2)-producing growth. The actual ΔG values for the formate metabolism are calculated to range between -8 and -20 kJ mol(-1) under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.