A Multi-Enzyme Cascade Reaction for the Production of 2'3'-cGAMP.
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 ScADK, AjPPK2, and SmPPK2 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.
['Becker|Martin|M|', 'Nikel|Patrick|P|', 'Andexer|Jennifer N|JN|', 'Lütz|Stephan|S|0000-0001-8534-0554', 'Rosenthal|Katrin|K|0000-0002-6176-6224']
D000150:Acinetobacter D000227:Adenine Nucleotides D000248:Adenosine Kinase D001426:Bacterial Proteins D055162:Biocatalysis D001709:Biotechnology D009712:Nucleotides, Cyclic D017856:Phosphotransferases (Phosphate Group Acceptor) D012441:Saccharomyces cerevisiae D029701:Saccharomyces cerevisiae Proteins D016962:Sinorhizobium meliloti
ATP cGAS enzyme cascade 2′3′-cGAMP biocatalysis cyclic dinucleotide (CDN) in vitro biotransformation multi-step reaction polyphosphate kinase