Purification, Cloning, and Sequencing of Archaebacterial Pyrophosphatase from the Extreme Thermoacidophile Sulfolobus acidocaldarius

Meyer, W.; Moll, Ralf; Kath, Thomas; Schäfer, Günter

doi:10.1006/abbi.1995.1277

Cited by 24 publications

(18 citation statements)

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“…(40)(41)(42)(43)(44), Methanothermobacter thermautotrophicus (Methanobacterium thermoautotrophicum) (45), and Thermococcus thioreducens (46) (Fig. 2A).…”

Section: Resultsmentioning

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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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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“…(40)(41)(42)(43)(44), Methanothermobacter thermautotrophicus (Methanobacterium thermoautotrophicum) (45), and Thermococcus thioreducens (46) (Fig. 2A).…”

Section: Resultsmentioning

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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“…Prokaryotic PPases are believed to be homohexamers, or tetramers, with a molecular weight per monomer of about 20 kDa, while eukaryotic enzymes are homodimers of about 30 kDa per monomer~Coop-erman et al, 1992!. The known archaebacterial pyrophosphatases resemble the eubacterial enzymes with respect to the molecular weight of a single subunit~Richter & Schäfer, 1992;Wakagi et al, 1992;Meyer et al, 1995! although their oligomeric structure has been unclear.…”

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confidence: 99%

Sulfolobus acidocaldarius inorganic pyrophosphatase: Structure, thermostability, and effect of metal ion in an archael pyrophosphatase

et al. 1999

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The first crystal structure of an inorganic pyrophosphatase~S-PPase! from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 Å. S-PPase is a D 3 homohexameric protein with one Mg 2ϩ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases~PPase!. In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg 2ϩ , while only Asp65 and Asp102 do in S-PPase, and the Mg 2ϩ moves by 0.7 Å. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion.The monomer S-PPase structure is very similar to that of Thermus thermophilus~T-PPase! and Escherichia colĩ E-PPase!, root-mean-square deviations around 1 Å0Ca. But the hexamer structures of S-and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E-and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.

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“…Only PPases from a methanogenic and two thermophilic archaea have lower optimal pH (6.5-8) (Van Alebeek et al 1994;Jetten et al 1992;Richter and Schäfer 1992;Meyer et al 1995).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

Oliva

Romero

Ayala

et al. 2000

Archives of Microbiology

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A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).

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Purification, Cloning, and Sequencing of Archaebacterial Pyrophosphatase from the Extreme Thermoacidophile Sulfolobus acidocaldarius

Cited by 24 publications

References 0 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

Sulfolobus acidocaldarius inorganic pyrophosphatase: Structure, thermostability, and effect of metal ion in an archael pyrophosphatase

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

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