Spotlight pubs.acs.org/acschemicalbiology
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ARTIFICIAL METALLOENZYMES ENABLE IN VIVO OLEFIN METATHESIS
their technology may become a useful new member of the bioorthogonal chemistry toolkit. Heidi A. Dahlmann
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GENETICALLY RECODED ORGANISM UNDER CONSTRUCTION
Adapted by permission from Macmillan Publishers Ltd.: Nature, advance online publication, 29 August 2016, DOI: 10.1038/ nature19114.
Transition metal-catalyzed reactions such as olefin metathesis, which joins C−C double bonds together end-to-end, are workhorse reactions in industry, but they often require environmentally unfriendly or toxic organic solvents. Modern process chemistry puts an emphasis on carrying out chemical transformations under “green” conditions, for example by using water as a solvent. Although transition metal catalysts often perform poorly in aqueous solutions, reactions mediated by these catalysts have been enabled by the development of water-soluble catalysts and detergents that create hydrophobic reaction environments. In a recent report, researchers in the laboratories of Sven Panke and Thomas R. Ward describe how they have taken this concept a few steps farther by engineering a metalloenzyme to carry out olefin metathesis in cells (Nature 2016, DOI: 10.1038/ nature19114). The research team chose streptavidin as the protein to host the ruthenium catalyst needed for carrying out olefin metathesis; furthermore, they engineered E. coli to express streptavidin within the periplasm rather than cytoplasm of the bacteria to avoid high cytoplasmic concentrations of the catalyst-poisoning biological reducing agent glutathione. To lure the catalyst into the protein, they tethered it to biotin, a small molecule with an extremely high affinity for streptavidin. Upon exposing this “biot-Ru” to cells containing periplasmic streptavidin, biot-Ru became incorporated into the streptavidin to form an active “metathase” capable of catalyzing metathesis reactions. The development of a metathase that was functional in cells was an important breakthrough; previously developed metathases had to be isolated before application in aqueous suspensions, which required very time-consuming purification. In contrast, hosting a functional metathase within living cells allowed the research team to screen streptavidin mutants for greater activity and use directed evolution to optimize metathases for specific substrates. The authors anticipate that © 2016 American Chemical Society
Image from N. Ostrov et al., Science, 2016, 353, 819. Reprinted with permission from AAAS.
Image from N. Ostrov et al., Science, 2016, 353, 819. Reprinted with permission from AAAS.
Preliminary results of an attempt to recode an organism’s entire genome are in, as reported by George M. Church and co-workers at Harvard University (Science 2016, 353, 819). The research team hopes to produce a genomically recoded organism with synthetic biological features useful for industrial applications. In order for an organism to grow and survive, its genomic information is transcribed from DNA into RNA, which in turn is translated into proteins. During translation, three-nucleotide segments of the RNA strand, or codons, are matched to complementary tRNA molecules charged with specific amino acids. Among the 64 possible codons, many codons are redundant, meaning that multiple codons exist for specifying the same amino acid or translation termination signal. Church and co-workers used a computer program to scan a simplified E. coli genome for all instances of seven codons with redundant functions and design a genome in which each of these codons was replaced with a synonymous alternative, reducing the number of codons to 57. To test whether the recoded genome would be viable, the research team synthesized it in 87 fragments that were incorporated individually into E. coli strains in which the corresponding segments of genomic DNA were removed. Published: September 16, 2016 2387
DOI: 10.1021/acschembio.6b00780 ACS Chem. Biol. 2016, 11, 2387−2389
ACS Chemical Biology
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After two dozen rounds of evolution, a ribozyme containing 17 mutations relative to the wild-type ribozyme (designated 24−3) was isolated. Ribozyme 24−3 was able to extend primers through sequences that halted the wild-type ribozyme, including past purine-rich regions and short stem-loop structures. Impressively, 24−3 also catalyzed the synthesis of yeast phenylalanyl tRNA from a 15-mer primer and, in a feat unprecedented for a synthetic ribozyme, exponentially amplified an RNA substrate in a PCR-like reaction. If the ribozyme could be further evolved to replicate itself, the authors note, then it would fulfill the criteria for classification as a synthetic form of RNA life.
At the time of their report, Church and co-workers had checked 55 segments of recoded genome; so far, 99.5% of all genes and greater than 90% of essential recoded genes supported cell viability and only 13 lethal recoding modifications were identified. They also developed a pipeline to efficiently identify and overcome these design flaws and demonstrated it on one example. Encouraged by these results, the authors anticipate assembling recoded organisms suitable for biotech applications; the organisms would resist viral infection and horizontal gene transfer and could possibly be engineered so that the seven stripped-out codons could be reassigned to non-natural amino acids. Heidi A. Dahlmann
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Heidi A. Dahlmann
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POLYMERASE RIBOZYME SYNTHESIZES COMPLEX RNA MOLECULES
AT THE HEART, A HISTIDINE
Adapted with permission from Green, A. P., et al. J. Am. Chem. Soc., 138, 11344−11352. Copyright 2016 American Chemical Society.
Adapted with permission from Green, A. P., et al. J. Am. Chem. Soc., 138, 11344−11352. Copyright 2016 American Chemical Society.
Protein enzyme active sites harbor intricate interactions between structurally adjacent amino acids utilizing both the peptide backbone and side chains. Precise positioning is critical for substrate binding and efficient catalysis, so slight alterations in this geometry can have profound effects. A frequent player on the front lines of catalysis is histidine due to its malleable chemical role. In the case of heme peroxidases, histidine interacts with both a heme-iron and an aspartate side chain at the active site. Mutagenesis of active site amino acids helps dissect the mechanism of enzymes, but recent advances in non-natural amino acid incorporation allow researchers to go a step further and perform atomic mutagenesis. Green et al. (J. Am. Chem. Soc. 2016 DOI: 10.1021/ jacs.6b07029) set out to examine the importance of histidine-aspartate pairing in one heme enzyme, ascorbate peroxidase (APX2), by using this fine grained technique. In this case, the mutant peroxidase enzyme had Nδ-methyl histidine (NMH) in place of the critical histidine. The presence of NMH in place of histidine meant that a putatively essential hydrogen bond to the conserved aspartate was disrupted by a methyl group. It also alters the chemical properties of the imidazole ring and locks it into one tautomeric form. Interestingly, the net result was a peroxidase enzyme that formed no interaction with aspartate yet displayed remarkably improved catalytic properties. The APX2 NMH enzyme performed a significantly higher number of turnovers on a model substrate and was more tolerant to changes at the conserved aspartate position. This study unveils new information on a long studied class of enzymes,
Horning, D. P., and Joyce, G. F. Proc. Natl. Acad. Sci., U.S.A., 113, 9786−9791. Copyright 2016 National Academy of Sciences, U.S.A.
Horning, D. P., and Joyce, G. F. Proc. Natl. Acad. Sci., U.S.A., 113, 9786−9791. Copyright 2016 National Academy of Sciences, U.S.A.
Amplification of functional RNA by a polymerase consisting entirely of RNA, a critical breakthrough in the field of evolutionary biology, was recently reported by researchers at the Scripps Research Institute (Proc. Natl. Acad. Sci., U.S.A. 2016, 113, 9786−9791). In their article, coauthors David P. Horning and Gerald F. Joyce describe the optimization of an RNA-based RNA polymerase to synthesize RNAs with complex structures including aptamers, other ribozymes, and tRNA. Modern organisms replicate themselves using protein-based polymerases to copy their DNA, the molecule that transmits genetic information from one generation to the next. However, scientists hypothesize that in a prebiotic environment lacking proteins, genetic information was stored in RNA that was replicated by RNA itselfspecifically by catalytically active RNA structures known as ribozymes. Horning and Joyce began with the previously discovered class I RNA polymerase ribozyme, first engineering it to contain structural features known to improve its catalytic function. The research duo then performed successive rounds of in vitro evolution, selecting for mutant polymerases that accurately extended primers to create full length aptamers that bound to immobilized target ligands. 2388
DOI: 10.1021/acschembio.6b00780 ACS Chem. Biol. 2016, 11, 2387−2389
ACS Chemical Biology
Spotlight
the peroxidases, and shows a potential for non-natural amino acids in optimizing catalysis. Jason G. Underwood
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TARGETING ZIKA Linked to devastating birth defects including microcephaly, Zika virus now looms as a major global health threat. No vaccines or treatments are currently available, and researchers are scrambling to find ways to treat or prevent infections. Toward that goal, Zhao et al. report the isolation and structural characterization of antibodies that protect mice against Zika infection (Cell, 2016, 166, 1016−1027). Previously, researchers had shown that neutralizing antibodies can target flavivirusesthe class that includes Zika and dengue feverby zeroing in on regions in their envelope (E) protein. Antibodies that bind to the DIII region of the E protein of a particular virus tend to be type-specific. So Zhao et al. used immunocompromised mice infected with strains of Zika virus to produce 2000 hybridomas. After screening, they isolated six monoclonal antibodies (mAbs) that bound to the E protein of Zika. Five mAbs were specific for Zika virus, and four of them neutralized Zika infection in cells. The team then looked for binding to the DIII region of the E protein, by measuring the affinity of all six for a recombinant DIII protein expressed in E. coli and using a functional assay. They then used X-ray crystallography to probe where four antibody fragments bound to the DIII protein. With docking and mapping studies, they examined these areas of the protein and their accessibility on the surface of the whole virus. Two of the DIII binding sites are not accessible on the surface of the whole virus, but ZV-67, for example, binds to the lateral ridge (LR), which is accessible on the virion surface. In mice, the two antibodies that bind to the LR region, ZV-54 and ZV-67, reduced viral load and protected the animals against a lethal strain of the Zika virus. This study provides a basis for understanding how antibodies can neutralize Zika virus. Though it is unclear whether the same surface regions also serve as epitopes in human infections, they could be useful as diagnostics or as a first step toward therapies or vaccines. Sarah A. Webb
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DOI: 10.1021/acschembio.6b00780 ACS Chem. Biol. 2016, 11, 2387−2389
