Colorimetric Determination of Pyrophosphate Anion and Its Application to Adenylation Enzyme Assay

Katano, Hajime; Watanabe, Hiroyuki; Takakuwa, Masahiro; Maruyama, Chitose; Hamano, Yoshimitsu

doi:10.2116/analsci.29.1095

Cited by 23 publications

(16 citation statements)

References 11 publications

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“…This information is essential for the determination of the target and method for engineering. In case of the substrate specificity and affinity assays, integrated UV assay with NADPH consumption for acyltransferase activity in PKS (Lowry et al, 2013), and pyrophosphate exchange assay, pyrophosphate release assay, and hydroxylamine quenching assay for adenylation activity in NRPS have been performed (Villiers and Hollfelder, 2009;Wilson and Aldrich, 2010;Katano et al, 2013;Stanisic and Kries, 2019). The substrate specificity of AT, A, KS, and C domains in the elongation module toward growing polyketide chains or polypeptide tethered to phosphopantetheine (Ppant) arm of ACP and T domain was studied by using the mimicking molecule such as acyl-or aminoacyl-N-acetylcysteamine thioesters (acyl-or aminoacyl-SNACs) (Ehmann et al, 2000;Jensen et al, 2012).…”

Section: Design Toolsmentioning

confidence: 99%

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Hwang

Lee

Cho

et al. 2020

Front. Mol. Biosci.

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In nature, various enzymes govern diverse biochemical reactions through their specific three-dimensional structures, which have been harnessed to produce many useful bioactive compounds including clinical agents and commodity chemicals. Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are particularly unique multifunctional enzymes that display modular organization. Individual modules incorporate their own specific substrates and collaborate to assemble complex polyketides or non-ribosomal polypeptides in a linear fashion. Due to the modular properties of PKSs and NRPSs, they have been attractive rational engineering targets for novel chemical production through the predictable modification of each moiety of the complex chemical through engineering of the cognate module. Thus, individual reactions of each module could be separated as a retro-biosynthetic biopart and repurposed to new biosynthetic pathways for the production of biofuels or commodity chemicals. Despite these potentials, repurposing attempts have often failed owing to impaired catalytic activity or the production of unintended products due to incompatible protein-protein interactions between the modules and structural perturbation of the enzyme. Recent advances in the structural, computational, and synthetic tools provide more opportunities for successful repurposing. In this review, we focused on the representative strategies and examples for the repurposing of modular PKSs and NRPSs, along with their advantages and current limitations. Thereafter, synthetic biology tools and perspectives were suggested for potential further advancement, including the rational and large-scale high-throughput approaches. Ultimately, the potential diverse reactions from modular PKSs and NRPSs would be leveraged to expand the reservoir of useful chemicals.

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Section: Design Toolsmentioning

confidence: 99%

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Hwang

Lee

Cho

et al. 2020

Front. Mol. Biosci.

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“…Pyrophosphate can be detected directly through the colored molybdopyrophosphate complex, however this method suffers from poor sensitivity . Alternatively, pyrophosphatase converts PP i into orthophosphate which forms a colored complex with molybdate and malachite green, which is feasible in a microplate format .…”

Section: Kinetic Profilingmentioning

confidence: 99%

Adenylation Domains in Nonribosomal Peptide Engineering

Stanišić

Kries

2019

ChemBioChem

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Nonribosomal peptides are a prolific source of bioactive molecules biosynthesized on large, modular assembly line synthetases. Synthetic biologists seek to obtain tailored peptides with tuned or novel bioactivities by engineering modules and domains of these nonribosomal peptide synthetases. The activation step catalyzed by adenylation domains primarily selects which amino acids are incorporated into nonribosomal peptides. Here, we review experimental protocols for probing the adenylation reaction that are applicable in natural product discovery and engineering. Several alternatives to the established pyrophosphate exchange assay will be compared and potential pitfalls pointed out. Binding pocket mutagenesis of adenylation domains has been successfully conducted to adjust substrate preferences. Novel screening methods relying on yeast surface display, for instance, search a larger sequence space for improved mutants and thus allow more substantial changes in peptide structure.

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“…7). 10,11 First, a 20-μL aliquot of the sample solution is mixed with 200 μL of the 20 mM Na2MoO4, 0.6 M HCl, and 60% (v/v) acetonitrile (AN) solution to prepare an acetonitrile-water (AN-W) mixture. In this mixture, PPi forms a yellow 18- 33 The sample solution, that is, the enzyme reaction mixture would consist of ATP and AMP, as 4-anion is reduced to the 8-electron reduced species, 33 which renders the solution blue.…”

Section: ·3 Colorimetric Ppi Determination and Adenylation Enzyme Assaymentioning

confidence: 99%

“…Furthermore, we developed a colorimetric method for the determination of pyrophosphate (PPi) anion. 10,11 The PPi determination has been applied to the assay of adenylation enzyme, which plays an important role in the biosynthetic mechanism.…”

Section: Introductionmentioning

confidence: 99%

Analytical Methods for the Detection and Purification of ε-Poly-L-lysine for Studying Biopolymer Synthetases, and Bioelectroanalysis Methods for Its Functional Evaluation

et al. 2014

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Colorimetric Determination of Pyrophosphate Anion and Its Application to Adenylation Enzyme Assay

Cited by 23 publications

References 11 publications

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Adenylation Domains in Nonribosomal Peptide Engineering

Analytical Methods for the Detection and Purification of ε-Poly-L-lysine for Studying Biopolymer Synthetases, and Bioelectroanalysis Methods for Its Functional Evaluation

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