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

Abstract

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 undermore » 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). Altogether, 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.« less

Authors:
 [1];  [1];  [1]
  1. Univ. of Florida, Gainesville, FL (United States)
Publication Date:
Research Org.:
Univ. of Florida, Gainesville, FL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Chemical Sciences, Geosciences & Biosciences Division
OSTI Identifier:
1470721
Grant/Contract Number:  
FG02-05ER15650
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 2; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

McMillan, Lana J., Hepowit, Nathaniel L., and Maupin-Furlow, Julie A. Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents. United States: N. p., 2015. Web. doi:10.1128/AEM.03055-15.
McMillan, Lana J., Hepowit, Nathaniel L., & Maupin-Furlow, Julie A. Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents. United States. doi:10.1128/AEM.03055-15.
McMillan, Lana J., Hepowit, Nathaniel L., and Maupin-Furlow, Julie A. Fri . "Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents". United States. doi:10.1128/AEM.03055-15. https://www.osti.gov/servlets/purl/1470721.
@article{osti_1470721,
title = {Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents},
author = {McMillan, Lana J. and Hepowit, Nathaniel L. and Maupin-Furlow, Julie A.},
abstractNote = {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). Altogether, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.},
doi = {10.1128/AEM.03055-15},
journal = {Applied and Environmental Microbiology},
number = 2,
volume = 82,
place = {United States},
year = {2015},
month = {11}
}

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