Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii

Park, Sun Young; Lee, Bokhui; Park, Kwang-Su; Chong, Youhoon; Yoon, Moon-Young; Jeon, Sung-Jong; Kim, Dong-Eun

doi:10.1007/s00253-009-2314-z

Cited by 19 publications

(12 citation statements)

References 25 publications

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“…PPAs prevent the accumulation of PP i during DNA sequencing reactions (3,4), PCR (5), and single-base extension (SBE) reactions (6). PPAs are also used to increase the yields of RNA by in vitro transcription (7) and of GDP-modified sugars by enzymatic synthesis (8,9).…”

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confidence: 99%

“…PPAs of the class A type are not associated with the membrane and are widespread in all domains of life, including Archaea. Class A type PPAs of hyperthermophilic archaea are already used in PCR and DNA sequencing reactions (4,5). In contrast, PPAs with DHHA2 domains (IPR004097) and PPAs of the PP i -energized H ϩ pump (IPR004131), Mn 2ϩ -dependent (IPR022934), and PpaX-type (IPR023733) families are not as widely distributed in archaea and/or can be difficult to purify due to their association with membranes.…”

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confidence: 99%

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Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

McMillan

Hepowit

Maupin-Furlow

2016

Appl Environ Microbiol

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Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PP i ) to orthophosphate (P i ) are commonly used to accelerate and detect biosynthetic reactions that generate PP i as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PP i -hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a V max of 465 U · mg ؊1 for PP i hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PP i and Mg 2؉ . Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PP i by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PP i under conditions of reduced water activity that are a hurdle to current PPA-based technologies. Inorganic pyrophosphatases (PPAs) (EC 3.6.1.1) catalyze the hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate (PP i ) (P 2 O 7 4Ϫ ) to form 2 mol of orthophosphate (P i ) (PO 4 3Ϫ ) (1). PP i is a common by-product of metabolism, including the biosynthesis of DNA, RNA, protein, peptidoglycan, lipids (e.g., cholesterol), cellulose, starch, and other biopolymers (2). PP i is also formed during the posttranslational modification of proteins, including adenylation, uridylation, and ubiquitylation (2).The hydrolysis of PP i by PPA releases a considerable amount of energy (⌬G=°ϭ Ϫ19.2 kJ/mol) that can drive unfavorable biochemical transformations to completion. One example is in the synthesis of DNA by DNA polymerase. In this endergonic (⌬G=°ϭ ϩ2.1 kJ/mol) reaction, the 3=-hydroxyl group of the nucleotide that resides at the 3= end of the growing DNA strand serves as a nucleophile in the attack of the ␣ phosphorus of the incoming deoxynucleoside 5=-triphosphate (dNTP), thus releasing PP i (2). The polymerization of DNA is highly dependent on PPA to hydrolyze the energy-rich PP i to 2P i and to drive the synthesis reaction forward (2). Under standard conditions, DNA polymerase alone converts DNA to dNTPs.PPAs are used in a wide variety of biotechnology applications based on the ability of these enzymes to drive reactions f...

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confidence: 99%

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confidence: 99%

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

McMillan

Hepowit

Maupin-Furlow

2016

Appl Environ Microbiol

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“…Literature reports of increased PCR yield when a thermostable inorganic pyrophosphatase (IPPase) was included in the reactions inspired us to test this enzyme in a qPCR series to see if we could drive the reaction forward by degrading the pyrophosphate, one of the two products of the chain reaction [17], [18]. Unexpectedly, the addition of IPPase reduced the apparent reaction yield ( Figure 5A ).…”

Section: Resultsmentioning

confidence: 99%

Robust Quantification of Polymerase Chain Reactions Using Global Fitting

Carr

Moore

2012

PLoS ONE

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BackgroundQuantitative polymerase chain reactions (qPCR) are used to monitor relative changes in very small amounts of DNA. One drawback to qPCR is reproducibility: measuring the same sample multiple times can yield data that is so noisy that important differences can be dismissed. Numerous analytical methods have been employed that can extract the relative template abundance between samples. However, each method is sensitive to baseline assignment and to the unique shape profiles of individual reactions, which gives rise to increased variance stemming from the analytical procedure itself.Principal FindingsWe developed a simple mathematical model that accurately describes the entire PCR reaction profile using only two reaction variables that depict the maximum capacity of the reaction and feedback inhibition. This model allows quantification that is more accurate than existing methods and takes advantage of the brighter fluorescence signals from later cycles. Because the model describes the entire reaction, the influences of baseline adjustment errors, reaction efficiencies, template abundance, and signal loss per cycle could be formalized. We determined that the common cycle-threshold method of data analysis introduces unnecessary variance because of inappropriate baseline adjustments, a dynamic reaction efficiency, and also a reliance on data with a low signal-to-noise ratio.SignificanceUsing our model, fits to raw data can be used to determine template abundance with high precision, even when the data contains baseline and signal loss defects. This improvement reduces the time and cost associated with qPCR and should be applicable in a variety of academic, clinical, and biotechnological settings.

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“…PPase activity has been found to be associated with GTases of other RNA viruses such as Bluetongue virus and Avian reovirus [9,10] and thus the PPase activity may be an intrinsic activity of multifunctional L protein. The role Virus Genes of PPase activity in replication has been suggested across eukaryotic and prokaryotic species [11] and work on Pyrococcus horikoshii PPase showed that it enhances DNA synthesis with Taq polymerase in vitro [12]. Genomic colocalization of PPase and polymerase gene in Thermococcus onnurineus [13] and the activation of these genes by Fig.…”

Section: L-rtpase Domain Possesses Tripolyphosphatase Activitymentioning

confidence: 97%

The RNA triphosphatase domain of L protein of Rinderpest virus exhibits pyrophosphatase and tripolyphosphatase activities

Singh

Subbarao

2016

Virus Genes

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L protein of the Rinderpest virus, an archetypal paramyxovirus possesses RNA-dependent RNA polymerase activity which transcribes the genome into mRNAs as well as replicates the RNA genome. The protein also possesses RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase enzyme activities responsible for capping the mRNAs in a conventional pathway similar to that of the host pathway. Subsequent to the earlier characterization of the GTase activity of L protein and identification of the RTPase domain of the L protein, we report here, additional enzymatic activities associated with the RTPase domain. We have characterized the pyrophosphatase and tripolyphosphatase activities of the L-RTPase domain which are metal-dependent and proceed much faster than the RTPase activity. Interestingly, the mutant proteins E1645A and E1647A abrogated the pyrophosphatase and tripolyphosphatase significantly, indicating a strong overlap of the active sites of these activities with that of RTPase. We discuss the likely role of GTase-associated L protein pyrophosphatase in the polymerase function. We also discuss a possible biological role for the tripolyphosphatase activity hitherto considered insignificant for the viruses possessing such activity.

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Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii

Cited by 19 publications

References 25 publications

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

Robust Quantification of Polymerase Chain Reactions Using Global Fitting

The RNA triphosphatase domain of L protein of Rinderpest virus exhibits pyrophosphatase and tripolyphosphatase activities

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