Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein.

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a ß-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1-2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the ß-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.

Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.

Cell division in the archaeon Haloferax volcanii relies on two FtsZ proteins with distinct functions in division ring assembly and constriction.

In bacteria, the tubulin homologue FtsZ assembles a cytokinetic ring, termed the Z ring, and plays a key role in the machinery that constricts to divide the cells. Many archaea encode two FtsZ proteins from distinct families, FtsZ1 and FtsZ2, with previously unclear functions. Here, we show that Haloferax volcanii cannot divide properly without either or both FtsZ proteins, but DNA replication continues and cells proliferate in alternative ways, such as blebbing and fragmentation, via remarkable envelope plasticity. FtsZ1 and FtsZ2 colocalize to form the dynamic division ring. However, FtsZ1 can assemble rings independent of FtsZ2, and stabilizes FtsZ2 in the ring, whereas FtsZ2 functions primarily in the constriction mechanism. FtsZ1 also influenced cell shape, suggesting it forms a hub-like platform at midcell for the assembly of shape-related systems too. Both FtsZ1 and FtsZ2 are widespread in archaea with a single S-layer envelope, but archaea with a pseudomurein wall and division septum only have FtsZ1. FtsZ1 is therefore likely to provide a fundamental recruitment role in diverse archaea, and FtsZ2 is required for constriction of a flexible S-layer envelope, where an internal constriction force might dominate the division mechanism, in contrast with the single-FtsZ bacteria and archaea that divide primarily by wall ingrowth.

The nature of persistent interactions in two model ß-grasp proteins reveals the advantage of symmetry in stability.

Two proteins within the ß-grasp superfamily, the B1-domain of protein G and the small archaeal modifier protein 1, were investigated to elucidate the key determinants of structural stability at the level of individual interactions. These symmetrical proteins both contain two ß-hairpins which form a sheet flanked by a central α-helix. They were subjected to high temperature molecular dynamics simulations and the detailed behavior of each long-range interaction was characterized. The results revealed that in GB1 the most stable region was the C-terminal hairpin and in SAMP1 it was the opposite, the N-terminal hairpin. Experimental results for GB1 support this finding. In conclusion, it appears that the difference in the location and number of hydrophobic interactions dictate the differential stability which is accommodated due to structural symmetry of the ß-grasp fold. Thus, the hairpins are interchangeable and in nature this lends itself to adaptability and flexibility.

Halophilic to mesophilic adaptation of ubiquitin-like proteins.

Elucidating how proteins adapt from halophilic to mesophilic environments will enable a better understanding of protein evolution and folding. In this study, by directed evolution and site-directed mutagenesis of the halophilic ubiquitin-like protein (ULP) Samp2, we find that substitution of the prebiotic amino acid Asp31 by Gly is uniquely effective in the mesophilic adaptation of ULP. Sequence analysis shows that substitution of Asp/Glu in halophilic ULPs by Gly in mesophilic ULPs has higher occurrence than other substitutions, supporting the unique role of the substitution in the mesophilic adaptation of ULP. Molecular dynamic simulations indicate that the mesophilic adaptation might result from the effect of the substitution on the conformational flexibility of ULP.

Solution Structure and Dynamics of the Small Protein HVO_2922 from Haloferax volcanii.

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.

Rhodanese-Like Domain Protein UbaC and Its Role in Ubiquitin-Like Protein Modification and Sulfur Mobilization in Archaea.

Ubiquitin-like protein (Ubl) modification targets proteins for transient inactivation and/or proteasome-mediated degradation in archaea. Here the rhodanese-like domain (RHD) protein UbaC (HVO_1947) was found to copurify with the E1-like enzyme (UbaA) of the Ubl modification machinery in the archaeon Haloferax volcanii UbaC was shown to be important for Ubl ligation, particularly for the attachment of the Ubl SAMP2/3s to protein targets after exposure to oxidants (NaOCl, dimethyl sulfoxide [DMSO], and methionine sulfoxide [MetO]) and the proteasome inhibitor bortezomib. While UbaC was needed for ligation of the Ubl SAMP1 to MoaE (the large subunit of molybdopterin synthase), it was not important in the formation of oxidant-induced SAMP1 protein conjugates. Indicative of defects in sulfur relay, mutation of ubaC impaired molybdenum cofactor (Moco)-dependent DMSO reductase activity and cell survival at elevated temperature, suggesting a correlation with defects in the 2-thiolated state of wobble uridine tRNA. Overall, the archaeal stand-alone RHD UbaC has an important function in Ubl ligation and is associated with sulfur relay processes.IMPORTANCE Canonical E2 Ub/Ubl-conjugating enzymes are not conserved in the dual-function Ubl systems associated with protein modification and sulfur relay. Instead, the C-terminal RHDs of E1-RHD fusion proteins are the apparent E2 modules of these systems in eukaryotes. E1s that lack an RHD are common in archaea. Here we identified an RHD (UbaC) that serves as an apparent E2 analog with the E1-like UbaA in the dual-function Ubl sampylation system of archaea. Unlike the eukaryotic E1-RHD fusion, the archaeal RHD is a stand-alone protein. This new insight suggests that E1 function in Ubl pathways could be influenced by shifts in RHD abundance and/or competition with other protein partners in the cell.

Environmental factors influence the Haloferax volcanii S-layer protein structure.

S-layers commonly cover archaeal cell envelopes and are composed of proteins that self-assemble into a paracrystalline surface structure. Despite their detection in almost all archaea, there are few reports investigating the structural properties of these proteins, with no reports exploring this topic for halophilic S-layers. The objective of the present study was to investigate the secondary and tertiary organization of the Haloferax volcanii S-layer protein. Such investigations were performed using circular dichroism, fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The protein secondary structure is centered on ß-sheets and is affected by environmental pH, with higher disorder in more alkaline conditions. The pH can also affect the protein's tertiary structure, with higher tryptophan side-chain exposure to the medium under the same conditions. The concentrations of Na, Mg and Ca ions in the environment also affect the protein structures, with small changes in α-helix and ß-sheet content, as well as changes in tryptophan side chain exposure. These changes in turn influence the protein's functional properties, with cell envelope preparations revealing striking differences when in different salt conditions. Thermal denaturation assays revealed that the protein is stable. It has been reported that the S-layer protein N-glycosylation process is affected by external factors and the present study indicates for the first time changes in the protein structure.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems.

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.

Bacterioruberin extracts from a genetically modified hyperpigmented Haloferax volcanii strain: antioxidant activity and bioactive properties on sperm cells.

AIMS: To examine the antioxidant activity of Bacterioruberin (Bctr)-rich extracts isolated from a hyperpigmented, genetically modified Haloferax volcanii strain (HVLON3) and to investigate the effect on cold-sensitive ram sperm cells. METHODS AND RESULTS: The strain HVLON3 produces higher Bctr amounts than most haloarchaea (220 ± 13 mg g-1 DW). HVLON3-Bctr extract has higher antioxidant activity than ß-carotene (threefold) as evaluated using 2,2 diphenyl-1-picrylhydrazyl combined with Electron Paramagnetic Resonance analysis (EC50 4·5 × 10-5  mol l-1 vs 13·9 × 10-5  mol l-1 respectively). Different concentrations of HVLON3-Bctr extracts were assayed on ram sperm after freezing/thawing and physiologically relevant parameters were examined. Extracts containing 7 and 20 µmol l-1 Bctr significantly improved cell viability (P < 0·0001), total and progressive motility (P < 0·0001) and sperm velocities (P = 0·0172 for curvilinear velocity VCL, P = 0·0268 for average path velocity VAP and P = 0·0181 for straight line velocity VSL) and did not affect other parameters evaluated. CONCLUSIONS: HVLON3 is an excellent source of natural microbial C50 carotenoids with applicability in Biotechnology, Biomedical and Veterinary fields. HVLON3 Bctr extract improves the quality of cryopreserved ram sperm cells and could be applied to increase insemination yields. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an insight on the bioactive properties of a bioproduct derived from haloarchaea (carotenoids) which are so far underexploited.

The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

Haloferax volcanii Proteome Response to Deletion of a Rhomboid Protease Gene.

Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures.

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor.

A tRNA-derived fragment competes with mRNA for ribosome binding and regulates translation during stress.

Posttranscriptional processing of RNA molecules is a common strategy to enlarge the structural and functional repertoire of RNomes observed in all 3 domains of life. Fragmentation of RNA molecules of basically all functional classes has been reported to yield smaller non-protein coding RNAs (ncRNAs) that typically possess different roles compared with their parental transcripts. Here we show that a valine tRNA-derived fragment (Val-tRF) that is produced under certain stress conditions in the halophilic archaeon Haloferax volcanii is capable of binding to the small ribosomal subunit. As a consequence of Val-tRF binding mRNA is displaced from the initiation complex which results in global translation attenuation in vivo and in vitro. The fact that the archaeal Val-tRF also inhibits eukaryal as well as bacterial protein biosynthesis implies a functionally conserved mode of action. While tRFs and tRNA halves have been amply identified in recent RNA-seq project, Val-tRF described herein represents one of the first functionally characterized tRNA processing products to date.

Important roles for membrane lipids in haloarchaeal bioenergetics.

Recent advances in lipidomic analysis in combination with various physiological experiments set the stage for deciphering the structure-function of haloarchaeal membrane lipids. Here we focused primarily on changes in lipid composition of Haloferax volcanii, but also performed a comparative analysis with four other haloarchaeal species (Halobacterium salinarum, Halorubrum lacusprofundi, Halorubrum sodomense and Haloplanus natans) all representing distinctive cell morphologies and behaviors (i.e., rod shape vs. pleomorphic behavior). Common to all five haloarchaea, our data reveal an extraordinary high level of menaquinone, reaching up to 72% of the total lipids. This ubiquity suggests that menaquinones may function beyond their ordinary role as electron and proton transporter, acting simultaneously as ion permeability barriers and as powerful shield against oxidative stress. In addition, we aimed at understanding the role of cations interacting with the characteristic negatively charged surface of haloarchaeal membranes. We propose for instance that by bridging the negative charges of adjacent anionic phospholipids, Mg2+ acts as surrogate for cardiolipin, a molecule that is known to control curvature stress of membranes. This study further provides a bioenergetic perspective as to how haloarchaea evolved following oxygenation of Earth's atmosphere. The success of the aerobic lifestyle of haloarchaea includes multiple membrane-based strategies that successfully balance the need for a robust bilayer structure with the need for high rates of electron transport - collectively representing the molecular basis to inhabit hypersaline water bodies around the planet.

Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.

N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-ß1,4-GlcA-ß1,4-glucose-ß1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-ß-1,4-galacturonic acid-α1,4-GlcA-ß1,4-glucose-ß1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-ß-1,4-galacturonic acid-α1,4-GlcA-ß1,4-glucose-ß1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.

Proteome targets of ubiquitin-like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii.

Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known. Here, we expand the knowledge of proteins isopeptide linked to SAMP1. LC-MS/MS analysis of -Gly-Gly signatures derived from SAMP1 S85R conjugates cleaved with trypsin was used to detect sites of sampylation (23 lysine residues) that mapped to 11 target proteins. Many of the identified target proteins were associated with sulfur metabolism and oxidative stress including MoaE, SAMP-activating E1 enzyme (UbaA), methionine sulfoxide reductase homologs (MsrA and MsrB), and the Fe-S assembly protein SufB. Several proteins were found to have multiple sites of samp1ylation, and the isopeptide linkage at SAMP3 lysines (K18, K55, and K62) revealed hetero-SAMP chain topologies. Follow-up affinity purification of selected protein targets (UbaA and MoaE) confirmed the LC-MS/MS results. 3D homology modeling suggested sampy1ylation is autoregulatory in inhibiting the activity of its protein partners (UbaA and MoaE), while occurring on the surface of some protein targets, such as SufB and MsrA/B. Overall, we provide evidence that SAMP1 is a ubiquitin-like protein modifier that is relatively specific in tagging its protein partners as well as proteins associated with oxidative stress response.

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor.

The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L(-1). Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg gCDW (-1), respectively, at a maximum cell dry weight of 6.5 g L(-1). Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD650 of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

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

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 Vmax 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 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 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.

Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea.

Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease.