ATP regeneration by a single polyphosphate kinase powers multigram-scale aldehyde synthesis<i>in vitro</i>

Tavanti, Michele; Hosford, Joseph; Lloyd, Richard C.; Brown, Murray J. B.

doi:10.1039/d0gc03830j

Cited by 45 publications

(46 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, ADP phosphorylation can represent the rate‐determining step in ATP production from AMP mediated by bifunctional PPKs. Time course experiments of AMP phosphorylation with purified PPK2 enzymes showing delayed ATP synthesis compared to ADP would support this observation [35–36] . A wider comparison between PPK2s might help in understanding if a certain degree of activity and catalytic efficiency of PPK2‐III biocatalysts has been sacrificed to maintain a broader substrate scope.…”

Section: Polyphosphate Kinases‐2: Biochemical Featuresmentioning

confidence: 84%

“…If differences in protein preparation, reaction setup (divalent metal cation and its concentration, buffer, as well as polyP type and concentration) and ATP detection method (bioluminescence vs HPLC) can be neglected, then it can be speculated that the significantly difference in protein content for the two assays (Motomura=3 μg/mL vs Mordhorst=500 μg/mL) might have led to different results. Accordingly, exclusive ADP formation from AMP was observed when a panel of purified putative PPK2‐III enzymes was diluted above a certain threshold before testing [36] …”

Section: Polyphosphate Kinases‐2: Biochemical Featuresmentioning

confidence: 99%

See 1 more Smart Citation

Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

et al. 2021

Self Cite

View full text Add to dashboard Cite

Polyphosphate kinases (PPKs) have emerged as valuable candidates to address the unmet need for scalable recycling of the common enzyme cofactor adenosine‐5’‐triphosphate (ATP) because they use cheap, freely available and stable polyphosphate (polyP) salts as the phosphate donor. The aim of this review is not only to present recent efforts in the characterisation of PPKs but also to provide an overview of the challenges associated with their implementation in chemical manufacturing. In assessing the current status of polyP‐driven biocatalysts, we identify gaps that need to be filled for successful adoption of these biocatalysts in industry. Guidelines for a wider adoption of PPKs are also provided.

show abstract

Section: Polyphosphate Kinases‐2: Biochemical Featuresmentioning

confidence: 84%

Section: Polyphosphate Kinases‐2: Biochemical Featuresmentioning

confidence: 99%

Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The AjPPK2 and SmPPK2 were tested with substrate concentrations of 10 mM AMP and ADP, respectively, and 15 mM polyP calculated as single phosphate units. Assays were performed in 50 mM TRIS-HCl buffer containing 40 The ScADK had a specific activity of 4.3 ± 0.9 U mg −1 , which is substantially lower compared to the published values of about 100 U mg −1 [28]. The decreased specific activity might be caused by the His-tag, which was omitted in the published study.…”

Section: Specific Activities Of the Kinasesmentioning

confidence: 88%

“…Furthermore, another promising optimization of the enzyme cascade could be the use of class III PPK2 enzymes, which enable us to synthesize nucleoside 5 -triphosphates from nucleoside 5 -monophosphates by a single enzyme [38]. Recently, the limited list of characterized PPK2 enzymes has been expanded with more than 50 new enzymes that can be used for ATP regeneration from AMP [40]. One of these enzymes, a class III PPK2 of an unclassified Erysipelotrichaceae bacterium, was used for ATP regeneration during the synthesis of 4-methoxybenzaldehyde on a multigram scale with a carboxylic acid reductase.…”

Section: Adaptation Of the Assay Composition In The Multi-enzyme Cascade Reactionmentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

“…Several ATP regeneration systems have been described in literature including several polyphosphate kinase activities ( Andexer and Richter, 2015 ; Tavanti et al, 2021 ). Among the enzymes that were successfully used, was the thermostable PPK from Thermotoga maritima ( Honda et al, 2010 ; Kim and Zhang, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis

Usvalampi¹,

Li²,

Frey³

2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.

show abstract

ATP regeneration by a single polyphosphate kinase powers multigram-scale aldehyde synthesisin vitro

Abstract: Towards scalable ATP recycling: a newly identified PPK2-III biocatalyst unlocked fully in vitro multigram-scale aldehyde synthesis employing a carboxylic acid reductase.

Cited by 45 publications

References 68 publications

Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

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

Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis

Contact Info

Product

Resources

About