A synthetic biochemistry module for production of bio-based chemicals from glucose

April 11th, 2016 by Paul H Opgenorth

Nature Chemical Biology 12, 393 (2016). doi:10.1038/nchembio.2062

Authors: Paul H Opgenorth, Tyler P Korman & James U Bowie

Synthetic biochemistry, the cell-free production of biologically based chemicals, is a potentially high-yield, flexible alternative to in vivo metabolic engineering. To limit costs, cell-free systems must be designed to operate continuously with minimal addition of feedstock chemicals. We describe a robust, efficient synthetic glucose breakdown pathway and implement it for the production of bioplastic. The system's performance suggests that synthetic biochemistry has the potential to become a viable industrial alternative.

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Formation of a chloride-conducting state in the Maltose ATP-Binding Cassette (ABC) transporter [Protein Structure and Folding]

April 7th, 2016 by Carlson, M. L., Bao, H., Duong, F.

ABC transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations in order to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ABC transporter into a channel.

DNA Polymerase III, but not Polymerase IV, Must be Bound to {tau}-Containing DnaX Complex to Enable Exchange into Replication Forks [Enzymology]

April 7th, 2016 by Yuan, Q., Dohrmann, P. R., Sutton, M. D., McHenry, C. S.

Examples of dynamic polymerase exchange have been previously characterized in the model systems provided by coliphages T4 and T7. Using a dominant negative D403E Pol III α that can form initiation complexes and sequester primer termini, but not elongate, we investigated the possibility of exchange at the E. coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a containing DnaX complex, did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ, showed that τ1, τ2, or τ3 -DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size and gaps between fragments, possessed the ability to challenge pre-established replication forks and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.
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Decoding Polo-like kinase 1 signaling along the kinetochore–centromere axis

April 4th, 2016 by Robert F Lera

Nature Chemical Biology 12, 411 (2016). doi:10.1038/nchembio.2060

Authors: Robert F Lera, Gregory K Potts, Aussie Suzuki, James M Johnson, Edward D Salmon, Joshua J Coon & Mark E Burkard

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Raft-based interactions of gangliosides with a GPI-anchored receptor

April 4th, 2016 by Naoko Komura

Nature Chemical Biology 12, 402 (2016). doi:10.1038/nchembio.2059

Authors: Naoko Komura, Kenichi G N Suzuki, Hiromune Ando, Miku Konishi, Machi Koikeda, Akihiro Imamura, Rahul Chadda, Takahiro K Fujiwara, Hisae Tsuboi, Ren Sheng, Wonhwa Cho, Koichi Furukawa, Keiko Furukawa, Yoshio Yamauchi, Hideharu Ishida, Akihiro Kusumi & Makoto Kiso

Structural Divergence of the Group I Intron-Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases that Function in RNA Splicing [RNA]

April 1st, 2016 by Lamech, L. T., Saoji, M., Paukstelis, P. J., Lambowitz, A. M.

The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mt tRNATyr and promote the splicing of autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon-binding domains and that the catalytic domain uses a newly evolved group I intron-binding surface, which includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA-binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined X-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the A. fumigatus mtTyrRS showed that the overall topology of the group I intron-binding surface is conserved, but with variations in key intron-binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions that also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron-specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.
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Role of glyoxylate shunt in oxidative stress response [Microbiology]

April 1st, 2016 by Ahn, S., Jung, J., Jang, I.-A., Madsen, E. L., Park, W.

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the TCA cycle. The GS bypasses the carbon dioxide-producing steps of the TCA cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be upregulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including upregulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer: a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

Entry of Bluetongue virus capsid requires the late endosomal specific lipid lysobisphosphatidic acid [Lipids]

April 1st, 2016 by Patel, A., Mohl, B.-P., Roy, P.

The entry of viruses into host cells is one of the key processes for the infection.. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show utilizing an in vitro liposome penetration assay and cell biology that Bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosomal specific lipid lysobisphosphatidic acid (LBPA) for productive membrane penetration and viral entry. Further we provide preliminary evidence that LBPA facilitates pore expansion during membrane penetration suggesting a mechanism for lipid factor requirement of BTV. This data indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry.

Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling

March 28th, 2016 by Geoffrey A Smith

Nature Chemical Biology 12, 373 (2016). doi:10.1038/nchembio.2056

Authors: Geoffrey A Smith, Kenji Uchida, Arthur Weiss & Jack Taunton

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AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs

March 28th, 2016 by William H Parsons

Nature Chemical Biology 12, 367 (2016). doi:10.1038/nchembio.2051

Authors: William H Parsons, Matthew J Kolar, Siddhesh S Kamat, Armand B Cognetta III, Jonathan J Hulce, Enrique Saez, Barbara B Kahn, Alan Saghatelian & Benjamin F Cravatt

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