Viewpoint Cite This: Biochemistry 2019, 58, 2703−2705
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Installing Terminal-Alkyne Reactivity into Proteins in Engineered Bacteria Kathrin Lang* Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Lab for Synthetic Biochemistry, Technical University of Munich, Institute for Advanced Study, TUM-IAS, Lichtenberg Strasse 4, 85748 Garching, Germany
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the past 20 years have seen rapid progress in the development of so-called bioorthogonal chemistries, allowing us to chemoselectively label biomolecules in vitro and in cells.2 The paradigm example for a bioorthogonal reaction is the Cu(I)catalyzed azide−alkyne cycloaddition (CuAAC), often also termed the standard click reaction.3 Many different UAAs bearing either terminal-alkyne or azide functionalities have been introduced into proteins in a residue- or site-specific way using the approaches described above, and chemoselective reaction with the respective probes via click chemistry has led to distinct applications for enriching, visualizing, and identifying target proteins.2 All approaches have, however, in common the fact that the UAAs bearing alkyne or azide functionalities have to be synthesizedoften in laborious and expensive waysin a synthetic chemistry lab and added exogenously to the growing cell cultures. A new discovery from Michelle Chang’s lab at the University of California may make this step now obsolete. Writing in Nature, her team describes a biosynthetic pathway for the production of terminal-alkyne amino acids and the heterologous incorporation of one of these amino acids into the Escherichia coli proteome followed by selective labeling via CuAAC.4 Only very few natural products bear terminal-alkyne functionalities that can react with azides via CuAAC. The noncanonical amino acid β-ethynylserine [βes (Figure 2)], found in the soil bacterium Streptomyces cattleya, represents one such example; little however was known about how S. cattleya synthesizes this terminal-alkyne-bearing amino acid. Chang’s team tackled the challenge of identifying the responsible βes gene cluster with the vision to leverage the natural biosynthetic pathway for expanding the chemical diversity of proteins in heterologous hosts. By comparing genomes of bacteria that produce terminalalkyne amino acids such as βes and propargyl-glycine (Pra) to those that do not, the authors identified a putative βes biosynthetic gene cluster. The cluster encoding enzymes BesA−BesE was confirmed, and the biosynthetic pathway elucidated by individually knocking out each gene of the involved enzymes in the genetically tractable S. cattleya. Subsequently, comparative metabolomics was used to determine and analyze pathway intermediates. Furthermore, Chang and co-workers succeeded in reconstituting the pathway in vitro by expressing, purifying, and characterizing every enzyme of the βes pathway.
ature uses a limited set of 20 proteinogenic amino acids to build proteins. This somewhat narrow range of chemical functional groups allows proteins to perform a plethora of functions within living systems, from replicating DNA and catalyzing metabolic reactions to responding to stimuli and transporting molecules from one location to another, often however with the help of additional chemical moieties such as post-translational modifications and cofactors. In rare instances, organisms have evolved novel translational machinery components to incorporate either selenocysteine or pyrrolysine in response to nonsense codons, but commonly proteins are composed of the 20 natural amino acids. In recent years, it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties into proteins in bacteria and eukaryotes by reprogramming the genetic code and rewiring the translational machinery.1 Equipping proteins with functionalities beyond the ones provided by the 20 natural amino acids enables diverse applications, including approaches for imaging and probing proteins, for controlling protein activity, and for the design of new protein function and protein therapeutics.1,2 Unnatural amino acids (UAAs) can be incorporated into proteins in living cells in a residue- or site-specific fashion by using the cellular biosynthetic machinery. Residue-specific incorporation relies in its simplest form on the recognition of an amino acid analogue as a substrate for a natural amino acid’s aminoacyl tRNA synthetase (aaRS), leading to partial replacement of one of the 20 natural amino acidsas specified by the protein’s genewith an UAA. Via introduction of mutations into the amino acid binding pocket or the editing domain (the domain that is responsible for hydrolyzing improperly aminoacylated tRNAs), the range of substrates can be increased by UAAs that are not recognized by endogenous synthetases. Residue-specific introduction of designer amino acids allows generation of globally modified proteins with novel chemical and physical properties (Figure 1a). On the contrary, site-specific UAA incorporation methods rely on the use of an orthogonal aaRS that charges the UAA onto an orthogonal tRNA. This allows the specific incorporation of an UAA during mRNA translation in response to a prematurely introduced stop codon (most often the amber codon) placed at a user-defined site in a gene of interest. The site-specific functionalization of proteins with novel chemical moieties provides an important approach for the modification and temporal control of the molecular functions of proteins (Figure 1b). Concomitantly with significant developments in the ability to co-translationally incorporate UAAs into proteins in cells, © 2019 American Chemical Society
Received: May 1, 2019 Published: June 3, 2019 2703
DOI: 10.1021/acs.biochem.9b00392 Biochemistry 2019, 58, 2703−2705
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Biochemistry
Figure 1. Residue- and site-specific incorporation of UAAs into proteins using the cellular translational machinery.
Figure 2. Biosynthetic pathway to form alkyne-bearing amino acids Pra and βes and co-translational incorporation of Pra into proteins in E. coli and subsequent labeling via CuAAC.
The biosynthetic route to Pra and βes uncovered some unexpected and remarkable chemistry. Starting from free Llysine, biosynthesis of terminal-alkyne-bearing amino acids is initiated by radical chlorination of the γ-C of L-lysine, catalyzed by an α-ketoglutarate/Fe-dependent halogenase (BesD) to form 4-chloro-L-lysine (4-Cl-Lys). This unstable amino acid is subjected to an unusual oxidative C−C bond-cleaving reaction to form the vinyl halide amino acid, 4-chloro-allyl-glycine (4Cl-Alg), by the catalytic action of the oxidase BesC with concomitant release of formaldehyde and ammonia. The next enzyme in the cluster, BesB, a pyridoxal 5′-phosphate (PLP)dependent γ-lyase, is responsible for formation of the terminalalkyne moiety via elimination of chloride and formation of an allene intermediate that ultimately isomerizes to the final terminal-alkyne-bearing amino acid Pra. Biosynthesis of βes involves two further steps. First, Pra is ligated to glutamate via its α-amino group forming a Glu-Pra dipeptide (catalyzed by BesA). Glu-Pra forms the substrate for the last enzyme in the
βes biosynthetic cluster, the hydroxylase BesE, to yield the γglutamyl-L-βes dipeptide. Upon amide bond hydrolysis, βes is released. The biosynthetic pathway to form Pra could be reproduced in the heterologous host E. coli by overexpressing BesB, BesC, and BesD, and endogenously produced Pra could be introduced into proteins in E. coli in a residue-specific way by co-expressing an engineered aaRS (PraRS),5 as visualized by fluorescently labeling the E. coli proteome via CuAAC and further substantiated by proteomic analysis of cell extracts. With this work, the authors have generated a completely autonomous organism that encodes a novel terminal-alkynebearing amino acid that can be incorporated into proteins and allows downstream bioorthogonal chemistries for enriching, modifying, and visualizing translational products. 2704
DOI: 10.1021/acs.biochem.9b00392 Biochemistry 2019, 58, 2703−2705
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Biochemistry
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kathrin Lang: 0000-0002-1318-6567 Funding
Our lab is supported by the Excellence Initiative CIPSM and the DFG through the following programmes: GRK1721, SFB1309, SPP1623, and SFB1035. K.L. is a Mössbauer Professor at TUM-IAS and as such acknowledges funding by the Excellence Initiative and the EU Marie Curie COFUND Program. Notes
The author declares no competing financial interest.
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REFERENCES
(1) Chin, J. W. (2017) Expanding and reprogramming the genetic code. Nature 550, 53−60. (2) Lang, K., and Chin, J. W. (2014) Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764−806. (3) (a) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A stepwise huisgen cycloaddition process: copper(I)catalyzed regioselective ″ligation″ of azides and terminal alkynes. Angew. Chem., Int. Ed. 41, 2596−9. (b) Tornøe, C. W., Christensen, C., and Meldal, M. (2002) Peptidotriazoles on solid phase: [1,2,3]triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057−3064. (4) Marchand, J. A., Neugebauer, M. E., Ing, M. C., Lin, C. I., Pelton, J. G., and Chang, M. C. Y. (2019) Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 567, 420−424. (5) Truong, F., Yoo, T. H., Lampo, T. J., and Tirrell, D. A. (2012) Two-strain, cell-selective protein labeling in mixed bacterial cultures. J. Am. Chem. Soc. 134, 8551−6.
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DOI: 10.1021/acs.biochem.9b00392 Biochemistry 2019, 58, 2703−2705
