A Multi-Enzyme Cascade Reaction for the Production of 2'3'-cGAMP

Overview

Journal Biomolecules

Publisher MDPI

Specialties Biochemistry
Molecular Biology

Date 2021 Apr 30

PMID 33923845

Citations 11

Authors

Martin Becker

Patrick Nikel

Jennifer N Andexer

Stephan Lutz

Katrin Rosenthal

Affiliations

Soon will be listed here.

Abstract

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ADK, PPK2, and PPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2'3'-cGAMP) formation was successfully developed. The 2'3'-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2'3'-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2'3'-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.

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References

Mordhorst S, Siegrist J, Muller M, Richter M, Andexer J . Catalytic Alkylation Using a Cyclic S-Adenosylmethionine Regeneration System. Angew Chem Int Ed Engl. 2017; 56(14):4037-4041. DOI: 10.1002/anie.201611038. View

Lu X, Wu H, Ye J, Fan Y, Zhang H . Expression, purification, and characterization of recombinant Saccharomyces cerevisiae adenosine kinase. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003; 35(7):666-70. View

Resnick S, Zehnder A . In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A. Appl Environ Microbiol. 2000; 66(5):2045-51. PMC: 101452. DOI: 10.1128/AEM.66.5.2045-2051.2000. View

Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Rohl I . cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature. 2013; 498(7454):380-4. PMC: 4143541. DOI: 10.1038/nature12306. View

Gaffney B, Veliath E, Zhao J, Jones R . One-flask syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogues. Org Lett. 2010; 12(14):3269-71. PMC: 2905038. DOI: 10.1021/ol101236b. View

Zhang X, Cui X, Li Z . Characterization of Two Polyphosphate Kinase 2 Enzymes Used for ATP Synthesis. Appl Biochem Biotechnol. 2020; 191(2):881-892. DOI: 10.1007/s12010-019-03224-6. View

Sehl T, Hailes H, Ward J, Wardenga R, von Lieres E, Offermann H . Two steps in one pot: enzyme cascade for the synthesis of nor(pseudo)ephedrine from inexpensive starting materials. Angew Chem Int Ed Engl. 2013; 52(26):6772-5. DOI: 10.1002/anie.201300718. View

Sun L, Wu J, Du F, Chen X, Chen Z . Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2012; 339(6121):786-91. PMC: 3863629. DOI: 10.1126/science.1232458. View

Nocek B, Kochinyan S, Proudfoot M, Brown G, Evdokimova E, Osipiuk J . Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria. Proc Natl Acad Sci U S A. 2008; 105(46):17730-5. PMC: 2584756. DOI: 10.1073/pnas.0807563105. View

10.

Batten L, Parnell A, Wells N, Murch A, Oyston P, Roach P . Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis. Biosci Rep. 2015; 36(1):e00294. PMC: 4748334. DOI: 10.1042/BSR20150203. View

11.

Zhang X, Shi H, Wu J, Zhang X, Sun L, Chen C . Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell. 2013; 51(2):226-35. PMC: 3808999. DOI: 10.1016/j.molcel.2013.05.022. View

12.

Corrales L, Glickman L, McWhirter S, Kanne D, Sivick K, Katibah G . Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep. 2015; 11(7):1018-30. PMC: 4440852. DOI: 10.1016/j.celrep.2015.04.031. View

13.

Motomura K, Hirota R, Okada M, Ikeda T, Ishida T, Kuroda A . A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation. Appl Environ Microbiol. 2014; 80(8):2602-8. PMC: 3993171. DOI: 10.1128/AEM.03971-13. View

14.

Hall J, Ralph E, Shanker S, Wang H, Byrnes L, Horst R . The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition. Protein Sci. 2017; 26(12):2367-2380. PMC: 5699495. DOI: 10.1002/pro.3304. View

15.

Rosenthal K, Becker M, Rolf J, Siedentop R, Hillen M, Nett M . Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2'-3'-Linked Cyclic Dinucleotides. Chembiochem. 2020; 21(22):3225-3228. PMC: 7754487. DOI: 10.1002/cbic.202000433. View

16.

Fehlau M, Kaspar F, Hellendahl K, Schollmeyer J, Neubauer P, Wagner A . Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System. Front Bioeng Biotechnol. 2020; 8:854. PMC: 7438870. DOI: 10.3389/fbioe.2020.00854. View

17.

Meng L, Liu Y, Yin X, Zhou H, Wu J, Wu M . Effects of His-tag on Catalytic Activity and Enantioselectivity of Recombinant Transaminases. Appl Biochem Biotechnol. 2019; 190(3):880-895. DOI: 10.1007/s12010-019-03117-8. View

18.

Schrittwieser J, Velikogne S, Hall M, Kroutil W . Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules. Chem Rev. 2017; 118(1):270-348. DOI: 10.1021/acs.chemrev.7b00033. View

19.

Petchey M, Rowlinson B, Lloyd R, Fairlamb I, Grogan G . Biocatalytic Synthesis of Moclobemide Using the Amide Bond Synthetase McbA Coupled with an ATP Recycling System. ACS Catal. 2020; 10(8):4659-4663. PMC: 7171872. DOI: 10.1021/acscatal.0c00929. View

20.

Andexer J, Richter M . Emerging enzymes for ATP regeneration in biocatalytic processes. Chembiochem. 2015; 16(3):380-6. DOI: 10.1002/cbic.201402550. View