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A LANTIBIOTIC WITHOUT LANTHIONINE?
LIPOPOLYSACCHARIDE ANTIGEN DEVELOPED FOR ANTIBACTERIAL VACCINATION STRATEGY
Reprinted with permission from Huo, L., and van der Donk, W.A. J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b02513. Copyright 2016 American Chemical Society.
The rise of economical genome sequencing in the past decade has revealed new antibiotics from diverse species. One class, the lanthipeptides, are synthesized by the ribosome but are then matured and modified by coexpressed enzymes. The precursor peptides are usually clustered together in bacterial operons, allowing for guided discovery of new lanthipeptides in genomes. The mature peptides possess antimicrobial activity and get the name lantibiotics due to the characteristic thioether cross-link known as lanthionine. To form lanthionine, the precursor peptide must have a cysteine, but interestingly, bacterial gene clusters have recently been discovered which appear like lanthipeptide operons but with no cysteines in the precursor peptide. Now, Huo and van der Donk (J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b02513) investigate a predicted cluster in Bacillus cereus SJ1 that encodes three precursor peptides, where only one harbors a cysteine. By coexpressing the BsjA precursor peptides with members of the gene cluster and analyzing the products by mass spectrometry, the researchers characterized the biosynthetic pathway to novel antibiotic peptides. They show that serine and threonine residues on the precursor peptides are first dehydrated by BsjM; then a portion are reduced by the dehydrogenase, BsjJB. Investigating the stereochemistry of reduction uncovered a rare finding. Several D-amino acids are present in the mature peptides and are installed by BsjJB. Finally, two genes, BsjT and BsjP, are important for the cleavage and maturation. The resulting antibiotic peptides include one lanthipeptide and a nonlanthipeptide dubbed bicereucin. Bacterial culture tests show that bicereucin has potent antimicrobial activity on several antibiotic-resistant Gram-positive strains. This study demonstrates how genome mining that starts with sequence homology can sometimes reveal unforeseen insights about functional diversity. Jason G. Underwood © 2016 American Chemical Society
Adapted by permission from Macmillan Publishers Ltd.: Nat. Chem. Kong, L., et al., 8, 242, copyright 2016.
In this dawning era of bacterial resistance to traditional antibiotics, research teams are scrambling to develop new ways to combat pathogenic microbes. An emerging approach is the production of vaccines that prime the immune system to recognize nonmammalian cell wall sugars found in pathogenic bacteria. One potential epitope, the tetrasaccharide Hep2Kdo2, serves as the “inner core” of lipopolysaccharide complexes in several pathogenic Gram-negative strains including Neisseria meningitides, Pseudomonas aeruginosa, and Escherichia coli. Until recently, it had not been possible to exploit this epitope for vaccine development because it is normally shielded by covalently attached outer-core glycan chains (i.e., capsular polysaccharides, or CPS) that vary between bacterial species. A team of researchers led by Benjamin G. Davis has now reported the chemical synthesis of a protein-lipopolysaccharide conjugate that displays free Hep2Kdo2 (Nat. Chem. 2016, 8, 242). The research team took on the challenge of producing the sterically congested Hep2Kdo2 core by linking an electrophilic Hep−Hep disaccharide with a nucleophilic Kdo−Kdo disaccharide, both coupling partners having been derived from mannose subunits extended using pre- or postglycosylation homologation reactions. Hep2Kdo2 cores were then attached via amine linkers to mutant dipteria toxin carrier protein to form the glycoconjugate vaccine candidate. Upon determining that rabbits immunized with the glycoconjugate raised antibodies against the Hep2Kdo2 core, Davis and co-workers found that antibody-containing sera could kill bacteria strains in which the biosynthesis of CPS, which would prevent the antibodies from accessing the inner core Hep2Kdo2 tetrasaccharide, had been knocked out or Published: May 20, 2016 1156
DOI: 10.1021/acschembio.6b00395 ACS Chem. Biol. 2016, 11, 1156−1158
ACS Chemical Biology
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inhibited. Finally, the authors demonstrated that the serum from glycoconjugate-immunized rabbits, in the presence of a nontoxic CPS inhibitor, significantly reduced the attachment of E. coli to host blood cells, thus potentially halting bacterial invasion of host cells in its earliest stages. Heidi A. Dahlmann
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targeting of TDO as a potential treatment strategy for AD, PD, and HD. Heidi A. Dahlmann
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MODULATION OF TRYPTOPHAN METABOLISM IMPEDES NEURODEGENERATION
SELECTIVE CYSTEINE-TAGGING METHOD REVEALS PROTEIN S-ACYLATION PATTERNS
Reprinted from Percher, A. et al. Proc. Natl. Acad. Sci., U.S.A. 113, 4302. Copyright 2016 National Academy of Sciences, U.S.A.
The covalent attachment of fatty acids to cysteine residues, or S-acylation, has been implicated in regulating the hydrophobicity, stability, trafficking, and activity of various eukaryotic proteins. Researchers have dissected many of these regulatory pathways either by treating cells with chemical probes that mimic fatty acids, which allows fluorescent labeling or enrichment of metabolically labeled proteins, or by extracting endogenously S-acylated proteins and performing thioesterexchange reactions with biotinylated or resin-bound reagents, which allows for the selective capture of the S-acylated proteins by affinity purification. Neither of these approaches enables the direct analysis of unmodified versus S-acylated proteins or the number of S-acylated sites in proteins with multiple cysteine residues, prompting Percher and co-workers to develop a new method, dubbed acyl-PEG exchange (APE; Proc. Natl. Acad. Sci., U.S.A. 2016, 113, 4302). In APE, cell lysates are treated with tris(2-carboxyethyl)phosphine to reduce nonacylated cysteine side chains to free thiols, which are then capped with N-ethylmaleimide (NEM). The thioesters of endogenous S-acylated residues are then cleaved with hydroxylamine to expose free thiol groups that are subsequently alkylated with PEG-labeled NEM, which alters the mobility of labeled proteins in SDS/PAGE. Western blotting is then used to visualize proteins of interest and reveal both nonmodified and endogenously mono- and poly-S-acylated protein isoforms. The research team refined their method by tracking the well-characterized S-acylated proteins HRas and CANX, then applied APE to determine the sites and levels of S-acylation in the less-characterized protein IFITM3, which is involved in antiviral immune responses. The utility of APE should enable more quantitative analysis of this dynamic post-translational lipidation of proteins in diverse cell and tissue types. Heidi A. Dahlmann
Reprinted from Breda, C. et al. Proc. Natl. Acad. Sci., U.S.A. DOI: 10.1073/pnas.1604453113. Copyright 2016 National Academy of Sciences, U.S.A.
Neurodegeneration, the process by which cells responsible for transmitting electrical or chemical signals progressively lose their structure or function, can have several possible manifestations. Familiar examples include Alzheimer’s disease (AD), in which a loss of neurons in the cerebral cortex leads to memory loss; Parkinson’s disease (PD), in which death of dopamine-secreting cells in the midbrain leads to difficulties with motor control; and Huntington’s disease (HD), in which gradual loss of brain cells beginning in the basal ganglia results in uncontrolled motions and dementia. Despite the diverse causes and effects of these diseases, they share certain pathophysiological traits such as perturbations in the degradation of tryptophan (TRP), a neurotransmitter precursor shown to have protective effects against neurodegeneration. In one particular TRP degradation pathway, the enzyme tryptophan-2,3-dioxygenase (TDO) mediates conversion of TRP to kynurenine, which can then either be converted to the neuroprotective kynurenic acid (KYNA) by kynurenine aminotransferases (KATs) or to the neurotoxic 3-hydroxykynurenine (3-HK) by kynurenine 3-monooxygenase (KMO). Increased production of neurotoxic metabolites in this pathway has been linked with several neurodegenerative disorders. A new report by Flaviano Giorgini and co-workers sheds light on the mechanisms by which TDO and KMO modulation can improve or worsen symptoms of neurodegeneration in AD, PD, and HD model fruit flies (Proc. Natl. Acad. Sci., U.S.A. 2016, DOI: 10.1073/pnas.1604453113). The research team demonstrated that downregulating KMO or TDO expression improved locomotor behavior in AD and PD fruit flies and reduced neurodegeneration in AD flies, effects presumably related to dramatically reduced 3-HK/KYNA ratios. The amelioration of neurodegeneration due to an improved 3-HK/KYNA ratio was also observed in HD flies in which TDO was knocked out. Meanwhile, pharmacological inhibition of TDO led to significant improvements in diseaserelevant phenotypes in all three disease models, supporting the 1157
DOI: 10.1021/acschembio.6b00395 ACS Chem. Biol. 2016, 11, 1156−1158
ACS Chemical Biology
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RADICAL SAM ENZYME PRIES OPEN RNA TARGET
From Schwalm, E. L., et al., Science 2016, 352, 309−312. Reprinted with permission from AAAS.
RlmN is an S-adenosylmethionine (SAM) enzyme, with an unusual mechanism, involving two SAM cofactors. It catalyzes the transfer of methyl groups to tRNA or rRNA. Most critically, it prompts the methylation of the C2 of a conserved adenosine in the peptidyltransferase region of rRNA, a modification that can confer antibiotic resistance in bacteria. Using X-ray crystallography, Schwalm et al. have now caught RlmN as it interacts with an RNA substrate (Science 2016, 352, 309−312). This 2.4 Å structure of the C118A variant of RlmN with tRNAGlu has several notable features. Both methionine and 5′-deoxyadenosine are visible in the structure, indicating that they remain in the active site until the methylated RNA is released from the binding pocket. This unusual catalyst binds to its substrate along the minor groove of the anticodon stem loop, primarily through interactions with the backbone sugars and phosphates. Anchored by a conserved Arg residue, it uses a critical β-strand as a lever to gain access to the unpaired but buried adenosine (A37) for methylation at the C2 position. The recognition strategy used here is different from RluA, the only other known enzyme that modifies two different types of RNA. Therefore, RlmN’s strategy may represent a new mechanism for dual specificity for tRNA and rRNA. In addition, this work highlights similarities in shape and structure between tRNAs and the bacterial ribosome core and demonstrates how a single enzyme can target substrates with different sequences but similar tertiary components. Sarah A. Webb
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DOI: 10.1021/acschembio.6b00395 ACS Chem. Biol. 2016, 11, 1156−1158
