Spotlight pubs.acs.org/acschemicalbiology
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DESIGNING INTEINS
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HYPOTHETICAL MINIMUM GENOME REACHES NEW LOW
From Hutchison C. A., III et al., Science, 2016, 351. DOI: 10.1126/science.aad6253. Reprinted with perission from AAAS.
Life can be complicated, even for the simplest of organisms, bacteria. Depending on their habitat, bacteria may need to scavenge for nutrients, synthesize their own amino acids, and produce antimicrobial defense molecules, in addition to maintaining cell growth and replication. In the absence of complicated demands, however, bacteria trim down their genomes in a “use it or lose it” manner. This has enabled parasitic species that colonize relatively stable niche environments to survive and thrive with astonishingly few genes; for example, Mycoplasma genitalium gets by with only 525 genes, the smallest known genome. The brevity of such genomes has made them a feasible target for synthesis in the laboratory; in 2010, researchers at the J. Craig Venter Institute (JCVI) chemically synthesized and installed a version of the Mycoplasma mycoides genome (JCVI-syn1.0) into bacterial cells (Science 2010, 329, 52). Inspired by the question of how few genes an organism would actually need to survive given a complete nutrient supply and an absence of selective pressure, Clyde A. Hutchison III and co-workers at JCVI have recently developed a functioning minimal bacterial genome (Science 2016, 351, aad6253). Their initial attempt to minimize JCVI-syn1.0 by deleting all genes that were putatively nonessential failed, in part because removing genes of proteins with redundant function left the transformed cells without any protein to carry out the necessary function. However, by subjecting JCVI-syn1.0 to multiple rounds of mutagenesis and screening the mutated genome portionwise to determine which genes were essential, nonessential, or simply necessary for robust growth, the team produced JCVI-syn3.0, a synthetic bacterium containing only 473 genes. Most of the retained genes were known to sustain cytosolic metabolism and cell membrane integrity or enable gene preservation or expression, but 79 of the genes could not be placed into a functional category, which opens the door for further genome optimization as the roles of these mystery genes are elucidated. Heidi A. Dahlmann
Reprinted with permission from Stevens et al., J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.5b13528. Copyright 2016 American Chemical Society.
Protein trans-splicing, a post-translational phenomenon, joins two separately expressed polypeptides through the activity of pendent split inteins in the cell. The two-piece nature of the reaction along with the convenience of self-processing has made split inteins a valuable tool for protein engineering and chemical biology approaches. Split inteins were first identified in cyanobacteria, and new family members have been uncovered by protein homology searches. Interestingly, despite their similar primary sequences, various family members display a wide range of activity with some splicing in seconds while others take hours. Now, Stevens et al. (J. Am. Chem. Soc. 2016, DOI: 10.1021/ jacs.5b13528) investigate the radical differences in splicing activity and use the data to build a better intein. They began with known “slow” and “fast” inteins encoded by different species but known to cross-react in splicing. These rate differences afforded a convenient system to carry out sequence comparisons or activity measurements with the two inteins and a series of chimeric constructs. A number of what the researchers term “accelerator” residues were identified around the active site for splicing. Next, sequence databases containing over 100 related inteins were queried and filtered for those containing the accelerator residues. Sequence comparisons of those filtered entries were used to guide creation of a new intein, Cfa, or consensus fast intein sequence. This designed intein showed remarkable activity with a rate exceeding the parental “fast” intein by 2.5 fold. Most remarkably, Cfa can splice at temperatures up to 80 °C and in high concentrations of denaturants such as urea or guanidine hydrochloride. They go on to show the utility of Cfa in the purification of insoluble proteins and in antibody conjugation in mammalian cells, two of the many possible uses for this promising new tool. Jason G. Underwood © 2016 American Chemical Society
Published: April 15, 2016 813
DOI: 10.1021/acschembio.6b00321 ACS Chem. Biol. 2016, 11, 813−815
ACS Chemical Biology
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REASSIGNING CODONS ENABLES RIOBOSOMAL INCORPORATION OF NON-PROTEINOGENIC AMINO ACIDS
Spotlight
C-NUCLEOSIDE CURBS EBOLA IN PRIMATES
Reprinted by permission from Macmillan Publishers Ltd.: Nature Warren et al., 531, 381−385. Copyright 2016.
Recently, the Ebola virus wreaked havoc on the people and healthcare systems of Sierra Leone, Liberia, and Guinea and remains a looming threat. In addition to the more than 11 000 deaths from this outbreak, survivors can experience additional health problems as the virus persists. So far, there are no fully tested antiviral drugs available to treat Ebola, but such a drug would be invaluable. Now, researchers have reported that a small molecule antiviral drug, GS-5734, suppresses viral activity and disease progression in primates infected with Ebola virus (Warren, T. K. et al., Nature 2016, 531, 381−385). GS-5734 is a nucleic acid analog, a C-nucleoside, that links to its adenine base via a carbon−carbon rather than a carbon− nitrogen bond. It also has a cyano group at the 1′ position of the ribose sugar. Previous research had shown that this class of molecules is active against RNA viruses. In vivo, this pro-drug is metabolized to an active triphosphate, which may work by curbing the activity of viral RNA-dependent RNA-polymerases. In cell-based assays, GS-5734 is selective, showing antiviral activity in cells infected with filoviruses related to Ebola, but not against retroviruses or other RNA viruses. To test this compound in vivo, the team used primates infected with clinically derived Ebola virus. After initial pharmacokinetic and drug distribution studies, the team conducted a two-part study to look at its effects. When 3 mg/kg of drug was given daily for 12 days starting either 0 days or 2 days after infection, viral load dropped and 28-day survival improved in both groups. In a second 12-day treatment study, the team optimized the dosing. The infected animals were divided into three groups: one received 10 mg/kg at day 2 followed by 3 mg/kg daily, another received 10 mg/kg at day 3 followed by 3 mg/kg daily, and a third received 10 mg/kg daily starting on day 3. All of the animals who started treatments on day 3 survived for the 28day study period, and those who received the highest doses showed the lowest viral loads. GS-5734 is the first small molecule drug reported that effectively treats Ebola in primates, and the drug is currently being evaluated for human safety in Phase I clinical trials. GS5734 could be manufactured in large quantities, making it a promising agent for testing against other similar viruses. Sarah A. Webb
Reprinted by permission from Macmillan Publishers Ltd.: Nat. Chem. Suga et al. 8, 317. Copyright 2016.
Researchers led by Hiroaki Suga at the University of Tokyo have recently co-opted the genetic code to develop a cell-free system in which nonproteinogenic amino acids (Naa) were incorporated by ribosomes into a peptide chain (Nat. Chem. 2016, 8, 317). The method opens up the possibility for circumventing chemical synthesis en route to developing libraries of peptides containing modified amino acids. In cells, protein synthesis is mediated by ribosomes, which catalyze the transfer of amino acids from tRNA (tRNA) molecules onto a growing peptide. The peptide amino acid sequence is dictated by the mRNA (mRNA) being translated. Each group of three consecutive nucleotides in the mRNA, or codon, matches a complementary anticodon in a corresponding tRNA. Although there are 64 possible combinations of codon nucleotides, they are genetically mapped to only 20 proteinogenic amino acids (Paa); consequently, some Paa, such as valine, glycine, and arginine, are redundantly assigned to four or six codons. In their recent work, Suga and co-workers artificially divided the redundant codon boxes to encode for two amino acids. For instance, GUG remained assigned to Val, and GUC was reassigned to an arbitrarily chosen Naa. Similarly, other redundant codon boxes could be reassigned to the cognate Paa and Naa. To demonstrate the utility of their new method, the research team carried out cell-free ribosome-mediated translation using tRNAs programmed according to the new genetic code, synthesizing a 32-mer peptide composed of 20 Paa and three Naa as well as a macrocyclic peptide containing nine Paa and four N-methylated Naa. Future applications may include the synthesis of libraries of nonstandard macrocyclic peptides for screening against drug targets. Heidi A. Dahlmann 814
DOI: 10.1021/acschembio.6b00321 ACS Chem. Biol. 2016, 11, 813−815
ACS Chemical Biology
Spotlight
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PET-DEGRADING BACTERIUM IDENTIFIED Polyethylene terephthalate (PET), prized for its durability and flexibility, has become ubiquitous worldwide in applications ranging from packaging to polyester clothing. Although PET is in principle recyclable, in practice most PET disposables end up discarded, and the chemical properties that make PET an ideal material for fabrication become liabilities in landfills or other sites of accumulation in the environment, such as waterways. Recognizing that microbes might be able to assist in breaking down PET into reusable substituents, thus reducing the need for further petroleum-based production of its monomers and serving as a potential environmental remediation strategy, Shosuke Yoshida and co-workers at multiple research institutes in Japan sought to discover microbes bacterial species that would be up to the task (Science 2016, 351, 1196). The research team screened 250 PET debris-contaminated samples collected from sediment, soil, wastewater, and sludge from PET-recycling siteto see if any samples could degrade low-crystallinity PET film; they found one microbial consortium that formed on the PET film that degraded the PET into carbon dioxide and water. From this consortium, a previously uncharacterized species of genus Ideonella that appeared to adhere to and damage the surface of the PET film substrate was isolated. The bacterium, which the research team proposed to name Ideonella sakaiensis, was found to nearly completely degrade the film after 6 weeks at 30 °C. Upon sequencing the bacterium’s genome and transcriptome, the researchers identified two enzymes, PET hydrolase (PETase) and MHETase, which were determined to catalyze sequential hydrolysis steps that break down PET into simpler substrates. These substrates can be further enzymatically converted to environmentally benign monomers that serve as a carbon source for growing I. sakaiensis. The authors speculate that the PET hydrolase may have evolved from a hydrolytic enzyme that breaks down cutin, a form of natural polyester produced in plants. Heidi A. Dahlmann
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DOI: 10.1021/acschembio.6b00321 ACS Chem. Biol. 2016, 11, 813−815
