Urea Increases Tolerance of Yeast Inorganic Pyrophosphatase Activity to Ethanol: The Other Side of Urea Interaction with Proteins

Lopes, Dahabada H. J.; Sola‐Penna, Mauro

doi:10.1006/abbi.2001.2529

Cited by 25 publications

(19 citation statements)

References 43 publications

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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.…”

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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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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…In particular, at low concentrations, ethanol retards the growth rate of yeasts and inhibits cell division, while high ethanol concentrations reduce cells viability and increase their death (Stanley et al, 2010). Furthermore, ethanol stress alters the metabolism (Hu et al, 2007) mostly acting on the plasma membrane and on the cytosolic enzymes (AnsanayGaleote et al, 2001, Lopes & Sola-Penna, 2001). Exposure to ethanol causes also a disruption of the membrane structure resulting in a loss of electrochemical gradients and transport associated to the membrane (D' Amore et al, 1990).…”

Section: Microbial Inhibition By Ethanolmentioning

confidence: 99%

“…Although this compound has long been considered as a protectant agent in the stress conditions (Ogawa et al, 2000) it was found that a mutant which accumulate high levels of trehalose showed a reduced expression of other adaptive mechanisms (Singer & Lindquist, 1998). According to Lopes & Sola-Penna (2001), the pyrophosphatase inactivation promoted by alcohols is not prevented by the presence of trehalose while 1.5 M urea attenuated this effect. It was established that during acclimatisation to ethanol stress, hundreds of genes are downregulated and about 100 genes are up-regulated (Alexandre et al, 2001;Chandler et al, 2004).…”

Section: Microbial Inhibition By Ethanolmentioning

confidence: 99%

Latest Frontiers in the Biotechnologies for Ethanol Production from Lignocellulosic Biomass

Paola¹,

Isabella²,

Romano³

2011

Biofuel Production-Recent Developments and Prospects

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Current Awareness

2002

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (3 weeks journals ‐ search completed 5th. Dec. 2001)

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Urea Increases Tolerance of Yeast Inorganic Pyrophosphatase Activity to Ethanol: The Other Side of Urea Interaction with Proteins

Cited by 25 publications

References 43 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

Latest Frontiers in the Biotechnologies for Ethanol Production from Lignocellulosic Biomass

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