Adenosine‐5′‐triphosphate‐dependent enzyme catalysed reactions are widespread in nature. Consequently, the enzymes involved have an intrinsic potential for use in syntheses of high value products. ...Although regeneration systems for ATP starting from adenosine‐5′‐diphosphate are available, certain limitations exist for both in vitro and in vivo applications requiring ATP regeneration from adenosine‐5′‐monophosphate, or adenosine. Following a short overview of the chemical and thermodynamic background, this Minireview focuses on emerging enzymes and methodologies for ATP regeneration. A large range of as yet unexploited reactions will be accessible with new, powerful, multistep ATP regeneration systems that use cheap phosphate donors and provide high longevity, compatibility, and robustness under process conditions. Their potential might go far beyond the direct use of ATP in enzymatic reactions; enzyme discovery, and engineering, as well as immobilisation strategies, will help to realise such systems.
New energy sources: The development of flexible and efficient multistep ATP regeneration systems could make a range of as yet unexploited biocatalytic reactions accessible for technical applications. Their potential might go far beyond the direct use of ATP in enzymatic reactions.
S‐Adenosylmethionine‐dependent methyltransferases are versatile tools for the specific alkylation of many compounds, such as pharmaceuticals, but their biocatalytic application is severely limited ...owing to the lack of a cofactor regeneration system. We report a biomimetic, polyphosphate‐based, cyclic cascade for methyltransferases. In addition to the substrate to be methylated, only methionine and polyphosphate have to be added in stoichiometric amounts. The system acts catalytically with respect to the cofactor precursor adenosine in methylation and ethylation reactions of selected substrates, as shown by HPLC analysis. Furthermore, 1H and 13C NMR measurements were performed to unequivocally identify methionine as the methyl donor and to gain insight into the selectivity of the reactions. This system constitutes a vital stage in the development of economical and environmentally friendly applications of methyltransferases.
Play it again, SAM: Biocatalytic alkylation is an emerging field in biotechnology. In addition to the discovery and engineering of highly efficient and selective methyltransferases, the development of an efficient regeneration system for the cofactor S‐adenosylmethionine (SAM) is a prerequisite for their economic application. The development of a biomimetic in vitro SAM regeneration cycle is presented with different methyltransferases as a model system.
Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are ...structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.
•Polyphosphate (polyP) sources differ significantly in their capability to provide reactive phosphate groups for the polyphosphate kinase-based ATP regeneration•The concentration of ATP and NADP+ in ...the in vitro reduction of carboxylic acids can be reduced to 200 μM and 100 μM, respectively•Lower NADP+ concentrations allow reducing the formation of alcohol from the aldehyde products through side activities in the enzyme preparations
Addressing the challenges associated with the development of in vitro biocatalytic carboxylate reductions for potential applications, important aspects of the co-factor regeneration systems and strategies for minimizing over-reduction were investigated. The ATP recycling can be performed with similarly high efficiency exploiting the polyphosphate source by combining Meiothermus ruber polyphosphate kinase and adenylate kinase or with Sinorhizobium meliloti polyphosphate kinase instead of the latter. Carboxylate reductions with the enzyme candidates used in this work allow operating at co-factor concentrations of adenosine 5′-triphosphate and β-nicotinamide adenine dinucleotide 2′-phosphate of 100 μM and, thereby, reducing the amounts of alcohols formed by side activities in the enzyme preparations. This study confirmed the expected benefits of carboxylic acid reductases in chemoselectively reducing the carboxylates to the corresponding aldehydes while leaving reductively-sensitive nitro, ester and cyano groups intact.
Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 ...(PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailedmechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.
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.
Mg2+‐dependent catechol‐O‐methyltransferases occur in animals as well as in bacteria, fungi and plants, often with a pronounced selectivity towards one of the substrate's hydroxyl groups. Here, we ...show that the bacterial MxSafC exhibits excellent regioselectivity for para as well as for meta methylation, depending on the substrate's characteristics. The crystal structure of MxSafC was solved in apo and in holo form. The structure complexed with a full set of substrates clearly illustrates the plasticity of the active site region. The awareness that a wide range of factors influences the regioselectivity will aid the further development of catechol‐O‐methyltransferases as well as other methyltransferases as selective and efficient biocatalysts for chemical synthesis.
Coenzyme A (CoA) is a common cofactor in biochemical reactions, and CoA‐dependent enzymes catalyze essential steps in anabolism and catabolism. This complex molecule also plays an important role in ...the synthesis of many high‐value products, such as synthetic antibiotics, vitamins, pheromones, and biopolymers. Nevertheless, the synthetic potential for biocatalytic processes cannot be fully exploited owing to the lack of an efficient regeneration system. Here, we report an acyl‐CoA regeneration system with integrated adenosine triphosphate (ATP) regeneration that is based on inexpensive polyphosphate as the single energy source. In the four‐enzyme cascade, two cofactors, acyl‐CoA and ATP, are each regenerated up to 2000 times. The applicability for different acyl donors and acceptors is shown by HPLC analysis. Owing to its flexibility toward virtually all relevant substrates, the system has the potential to make CoA‐dependent reactions more accessible for chemical synthesis in vitro.
Two in one: Inexpensive and stable polyphosphate fuels a four‐enzyme cascade for the application of acyl‐coenzyme A (acyl‐CoA)‐dependent transferases by using a broad range of acyl donors and acceptors. The two cofactors required in this system, acyl‐CoA and adenosine triphosphate (ATP), are regenerated by using carboxylate‐CoA ligases and polyphosphate kinases.
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