Inactivation of yeast inorganic pyrophosphatase by organic solvents

Grazinoli-Garrido, Rodrigo; Sola‐Penna, Mauro

doi:10.1590/s0001-37652004000400006

Cited by 5 publications

(6 citation statements)

References 20 publications

(26 reference statements)

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“…We demonstrate the use of HvPPA in a novel coupled assay to detect the PP i by-product released at reduced water activity (2 M NaCl) by ATP-dependent adenylation of the ubiquitin-like SAMP1 by the salt-loving E1-like enzyme UbaA. In contrast, current PPAs are inactivated in dose-dependent manner by salt and organic solvents (22)(23)(24). Our discovery of HvPPA and its function opens new possibilities for the hydrolysis of PP i and related compounds in systems which benefit from the use of high-ionicstrength compounds and/or organic solvents.…”

Section: Resultsmentioning

confidence: 99%

“…PPAs are used routinely to quantify rates of reactions that release PP i as a byproduct, such as single nucleotide polymorphism (SNP) genotyping reactions (10-12), RNA synthesis by viral RNA-dependent RNA polymerases (13), and aminoacyl-tRNA synthetase activity (14,15). The advantage of PPA-coupled assays is that PPA hydrolyzes PP i to a product (2P i ) which is readily detected by colorimetric assay (16,17).PPAs that operate in a wide variety of organic solvents and salt concentrations are desirable in bioindustry to increase the solubility of hydrophobic substrates, allow for novel synthetic chemistry, alter substrate specificity, ease product recovery, and reduce microbial contamination (18)(19)(20)(21)(22)(23)(24). Biotechnological applications of solvent-tolerant PPAs can be envisioned in the biosynthesis of hydrophobic compounds derived from carbon skeletons such as cholesterol and rubber.…”

mentioning

confidence: 99%

“…PPAs that operate in a wide variety of organic solvents and salt concentrations are desirable in bioindustry to increase the solubility of hydrophobic substrates, allow for novel synthetic chemistry, alter substrate specificity, ease product recovery, and reduce microbial contamination (18)(19)(20)(21)(22)(23)(24). Biotechnological applications of solvent-tolerant PPAs can be envisioned in the biosynthesis of hydrophobic compounds derived from carbon skeletons such as cholesterol and rubber.…”

mentioning

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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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

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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“…2). Interestingly, Grazinoli-Garrido and Sola-Penna (2004) have showed that the soluble yeast inorganic pyrophosphatase is also inactivated by several organic solvents (methanol, ethanol, propanol and acetone) depending on their hydrophobicity. This indicates that both the soluble and membrane bound pyrophosphatases seem to be highly sensitive to organic solvents.…”

Section: Discussionmentioning

confidence: 99%

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

Façanha

Okorokova‐Façanha

2008

Physiol Mol Biol Plants

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The electrochemical H + gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H + -pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H + -ATPase (V-ATPase) even in the absence of the H + gradient in a water-Me 2 SO co-solvent mixture, resulting in net synthesis of [γ-32 P]ATP from [ 32 P]P i and ADP. The water-Me 2 SO (5 to 20 %) media promoted inhibition of both PP i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH 4 + ). This effect of Me 2 SO on the ATP⇔ 32 P exchange reaction seems to be related to a decrease of the apparent K m of the V-ATPase for P i . The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.

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“…Most PPAs are inactive at high salt concentrations and organic solvents, thereby limiting their usage. PPAs that function in a wide range of organic solvents and salt concentrations are desirable in bioindustry to increase the solubility of hydrophobic substrates, which has advantages toward synthetic chemistry, to alter substrate specificity, ease product recovery, and reduce microbial contamination (Grazinoli‐Garrido & Sola‐Penna, 2004).…”

Section: Lignocellulosic Components and Depolymerizationmentioning

confidence: 99%

Halophilic archaea and their potential to generate renewable fuels and chemicals

Kasirajan

Maupin-Furlow

2020

Biotech & Bioengineering

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Lignocellulosic biofuels and chemicals have great potential to reduce our dependence on fossil fuels and mitigate air pollution by cutting down on greenhouse gas emissions. Chemical, thermal, and enzymatic processes are used to release the sugars from the lignocellulosic biomass for conversion to biofuels. These processes often operate at extreme pH conditions, high salt concentrations, and/or high temperature. These harsh treatments add to the cost of the biofuels, as most known biocatalysts do not operate under these conditions. To increase the economic feasibility of biofuel production, microorganisms that thrive in extreme conditions are considered as ideal resources to generate biofuels and value‐added products. Halophilic archaea (haloarchaea) are isolated from hypersaline ecosystems with high salt concentrations approaching saturation (1.5–5 M salt concentration) including environments with extremes in pH and/or temperature. The unique traits of haloarchaea and their enzymes that enable them to sustain catalytic activity in these environments make them attractive resources for use in bioconversion processes that must occur across a wide range of industrial conditions. Biocatalysts (enzymes) derived from haloarchaea occupy a unique niche in organic solvent, salt‐based, and detergent industries. This review focuses on the use of haloarchaea and their enzymes to develop and improve biofuel production. The review also highlights how haloarchaea produce value‐added products, such as antibiotics, carotenoids, and bioplastic precursors, and can do so using feedstocks considered “too salty” for most microbial processes including wastes from the olive‐mill, shell fish, and biodiesel industries.

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Inactivation of yeast inorganic pyrophosphatase by organic solvents

Cited by 5 publications

References 20 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

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

Halophilic archaea and their potential to generate renewable fuels and chemicals

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