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 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...

Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin‐like proteins of Archaea

“…Thus, UbaA was found to bind the nucleotide ligands AMP‐PNP, ATP, and ADP but not AMP, CTP, GTP, UTP, and TTP. These results are supported by our recent finding that PP i is released when UbaA is incubated with SAMPs and ATP, but not the other nucleoside triphosphates .…”

“…Previous work demonstrates that UbaA adenylates SAMPs (an early step in the pathway) . Here, we find that UbaA forms a thioester intermediate with the SAMPs (a later step in the pathway).…”

Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea

“…ATP was also shown to be important ( Fig. S4B , lanes 2 and 10), most likely due to the need for ATP to initiate sampylation through the E1 UbaA-mediated adenylation of the Ubl SAMP ( 14 , 28 ). DMS and DMSO 2 could not substitute for DMSO in stimulating the sampylation reaction ( Fig.…”

Haloferax volcanii UbaA, catalytic engine for sampylation and sulfur transfer

“…Surprisingly, AMP‐PNP (with nitrogen replacing oxygen between β‐ and γ‐phosphate) cannot substitute for ATP, making the reader wonder whether adenylation really involves a split between α‐ and β‐phosphate and the production of pyrophosphate. Any doubts, however, are eliminated by reference to earlier work and the observation that AMP‐PNP is a poor substrate of ubiquitin‐E1 too . Consistent with the chemistry of thioesters, the covalent SAMP UbaA complexes are (mostly) sensitive to reducing agents, and their formation can be altogether prevented by pretreatment of UbaA with N‐ethyl‐maleimide, a chemical that covalently modifies cysteine residues.…”

“…Both start with the conversion of the SAMPs to the corresponding adenylates, at the expense of ATP, and with concomitant production of pyrophosphate (Fig. A) . From there on, the pathways for protein conjugation and sulfur mobilization diverge.…”