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Enzymology and Protein Engineering

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

Lana J. McMillan, Nathaniel L. Hepowit, Julie A. Maupin-Furlow
M. J. Pettinari, Editor
Lana J. McMillan
aDepartment of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
bGenetics Institute, University of Florida, Gainesville, Florida, USA
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Nathaniel L. Hepowit
aDepartment of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
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Julie A. Maupin-Furlow
aDepartment of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
bGenetics Institute, University of Florida, Gainesville, Florida, USA
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M. J. Pettinari
Roles: Editor
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DOI: 10.1128/AEM.03055-15
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ABSTRACT

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi 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 PPi-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 PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg2+. 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 PPi 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 PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.

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Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents
Lana J. McMillan, Nathaniel L. Hepowit, Julie A. Maupin-Furlow
Applied and Environmental Microbiology Jan 2016, 82 (2) 538-548; DOI: 10.1128/AEM.03055-15

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Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents
Lana J. McMillan, Nathaniel L. Hepowit, Julie A. Maupin-Furlow
Applied and Environmental Microbiology Jan 2016, 82 (2) 538-548; DOI: 10.1128/AEM.03055-15
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