Structural Evolution of Bacterial Polyphosphate Degradation Enzyme for Phosphorus Cycling.

Living organisms ranging from bacteria to animals have developed their own ways to accumulate and store phosphate during evolution, in particular as the polyphosphate (polyP) granules in bacteria. Degradation of polyP into phosphate is involved in phosphorus cycling, and exopolyphosphatase (PPX) is the key enzyme for polyP degradation in bacteria. Thus, understanding the structure basis of PPX is crucial to reveal the polyP degradation mechanism. Here, it is found that PPX structure varies in the length of ɑ-helical interdomain linker (ɑ-linker) across various bacteria, which is negatively correlated with their enzymatic activity and thermostability - those with shorter ɑ-linkers demonstrate higher polyP degradation ability. Moreover, the artificial DrPPX mutants with shorter ɑ-linker tend to have more compact pockets for polyP binding and stronger subunit interactions, as well as higher enzymatic efficiency (kcat/Km) than that of DrPPX wild type. In Deinococcus-Thermus, the PPXs from thermophilic species possess a shorter ɑ-linker and retain their catalytic ability at high temperatures (70 °C), which may facilitate the thermophilic species to utilize polyP in high-temperature environments. These findings provide insights into the interdomain linker length-dependent evolution of PPXs, which shed light on enzymatic adaption for phosphorus cycling during natural evolution and rational design of enzyme.

Structure of the exopolyphosphatase (PPX) from Zymomonas mobilis reveals a two-magnesium-ions PPX.

Rapid adaptation of metabolic capabilities is crucial for bacterial survival in habitats with fluctuating nutrient availability. In such conditions, the bacterial stringent response is a central regulatory mechanism activated by nutrient starvation or other stressors. This response is primarily controlled by exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes. To gain further insight into these enzymes, the high-resolution crystal structure of PPX from Zymomonas mobilis (ZmPPX) was determined at 1.8 Å. The phosphatase activity of PPX was strictly dependent on the presence of divalent metal cations. Notably, the structure of ZmPPX revealed the presence of two magnesium ions in the active site center, which is atypical compared to other PPX structures where only one divalent ion is observed. ZmPPX exists as a dimer in solution and belongs to the "long" PPX group consisting of four domains. Remarkably, the dimer configuration exhibits a substantial and deep aqueduct with positive potential along its interface. This aqueduct appears to extend towards the active site region, suggesting that this positively charged aqueduct could potentially serve as a binding site for polyP.

The structure of exopolyphosphatase (PPX) from Porphyromonas gingivalis in complex with substrate analogs and magnesium ions reveals the basis for polyphosphate processivity.

The enzymes exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GppA) play important roles in the bacterial stringent response. PPX degrades inorganic polyphosphate (polyP), a polymer composed of a few to hundreds of phosphate residues supporting cell survival in the stationary phase. The crystal structure of PPX from Porphyromonas gingivalis (PgPPX) in complex with catalytic magnesium ions and several sulfate ions was solved. PgPPX contained two domains and represented a "closed" configuration. Four sulfate ions forming a linear dispersed chain were observed in the aqueduct of the PPX dimer, which the long polyP chain most likely occupied. The side chain of R255 stretched into the cavity where polyP could be located, obstructing the entrance of larger substrates such as NTP and NDP. This study provided the first view into the structure of the PPX/GppA homolog in complex with magnesium ions and substrate analogs and explained how PgPPX implemented its functionality.

An enzyme-based biosensor for monitoring and engineering protein stability in vivo.

Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.

New structural insights reveal an expanded reaction cycle for inositol pyrophosphate hydrolysis by human DIPP1.

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP-InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study, we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs ("PCP-InsPs"). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as "suspension cables." We demonstrate functionality for a triglycine peptide within a ß-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity.

Structural insights into DNA double-strand break signaling.

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.

Structural and functional study of SaAcP, an acylphosphatase from Staphylococcus aureus.

Acylphosphatase is the smallest enzyme that is widely distributed in many diverse organisms ranging from archaebacteria to higher-eukaryotes including the humans. The enzyme hydrolyzes the carboxyl-phosphate bonds of the acyl phosphates which are important intermediates in glycolysis, membrane pumps, tricarboxylic acid cycle, and urea biosynthesis. Despite its biological importance in critical cellular functions, very limited structural investigations have been conducted on bacterial acylphosphatases. Here, we first unveiled the crystal structure of SaAcP, an acylphosphatase from gram-positive S. aureus at the atomic level. Structural insights on the active site together with mutation study provided greater understanding of the catalytic mechanism of SaAcP as a bacterial acylphosphatase and as a putative apyrase. Furthermore, through NMR titration experiment of SaAcP in its solution state, the dynamics and the alterations of residues affected by the phosphate ion were validated. Our findings elucidate the structure-function relationship of acylphosphatases in gram-positive bacteria and will provide a valuable basis for researchers in the field related to bacterial acylphosphatases.

Functional Characterization of COG1713 (YqeK) as a Novel Diadenosine Tetraphosphate Hydrolase Family.

Diadenosine tetraphosphate (Ap4A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap4A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap4A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap4A in B. subtilis YqeK shows a catalytic efficiency toward Ap4A similar to that of the symmetrically cleaving Ap4A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.IMPORTANCE Elevation of Ap4A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap4A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap4A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae By identifying the player contributing to Ap4A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

Principles of RNA and nucleotide discrimination by the RNA processing enzyme RppH.

All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the Escherichia coli Nudix hydrolase RppH, which triggers 5'-end-dependent RNA degradation by removing orthophosphate from the 5'-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5'-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5'-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5'-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.

Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge.

The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

High resolution structure of Vibrio cholerae acylphosphatase (VcAcP) cage: Identification of drugs, location of its binding site and engineering to facilitate cage formation.

Protein cages have recently emerged as an extraordinary drug-delivery system due to its biocompatibility, biodegradability, low toxicity, ease to manipulate and engineer. We have reported earlier the formation and architecture of a do-decameric cage-like architecture of Vibrio cholerae acylphosphatase (VcAcP) at 3.1 Å. High resolution (2.4 Å) crystal structure of VcAcP cage, reported here, illuminates a potential binding site for sulphate/phosphate containing drugs whereas analysis of its subunit association and interfaces indicates high potential for cage engineering. Tryptophan quenching studies indeed discloses noteworthy binding with various sulphate/phosphate containing nucleotide-based drugs and vitamin B6 (PLP) demonstrating that exterior surface of VcAcP protein cage can be exploited as multifunctional carrier. Moreover, a quadruple mutant L30C/T68C/N40C/L81C-VcAcP (QM-VcAcP) capable to form an intricate disulphide bonded VcAcP cage has been designed. SEC, SDS-PAGE analysis and DLS experiment confirmed cysteine mediated engineered VcAcP cage formation.

Structure and activity of PPX/GppA homologs from Escherichia coli and Helicobacter pylori.

Rapid adaptation to environmental changes is crucial for bacterial survival. Almost all bacteria possess a conserved stringent response system to prompt transcriptional and metabolic responses toward stress. The adaptive process relies on alarmones, guanosine pentaphosphate (pppGpp), and tetraphosphate (ppGpp), to regulate global gene expression. The ppGpp is more potent than pppGpp in the regulatory activity, and pppGpp phosphohydrolase (GppA) plays a key role in (p)ppGpp homeostasis. Sharing a similar domain structure, GppA is indistinguishable from exopolyphosphatase (PPX), which mediates the metabolism of cellular inorganic polyphosphate. Here, our phylogenetic analysis of PPX/GppA homologs in bacteria shows a wide distribution with several distinct subfamilies, and our structural and functional analysis of Escherichia coli GppA and Helicobacter pylori PPX/GppA reveals unique properties of each homolog. These results explain how each homolog possesses its distinct functionality.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Enzymatic quantification and length determination of polyphosphate down to a chain length of two.

Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected. To overcome this analytical limitation, a method involving both the scPpx1p and the S. cerevisiae inorganic pyrophosphatase (scIpp1p) is proposed. Differential enzymatic hydrolysis of polyP with scPpx1p, and a combination of scIpp1p and scPpx1p allows not only for comprehensive quantification of polyP (excluding cyclic polyP) down to a chain length of two, but also absolute average chain length determination in the range of two to approximately 80. An optimized one-reagent method for rapid (2 min) orthophosphate quantification is part of the assay. Biological phosphorous containing molecules at equimolar phosphorous concentrations regarding polyP do not interfere. The method requires 1.5 µg polyP and calls only for a plate reader. This is the first enzymatic method for simultaneous average polyP chain length determination as well as comprehensive quantification.

Hydrolysis of diadenosine polyphosphates. Exploration of an additional role of Mycobacterium smegmatis MutT1.

Diadenosine polyphosphates (ApnA, n=2-6), particularly Ap4A, are involved in several important physiological processes. The substantial sequence identity of the Nudix hydrolase domain (domain 1) of Mycobacterium smegmatis MutT1 (MsMutT1) with a known Ap4A hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with Ap4A as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving Ap4A and Ap6A. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to ApnA were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with Ap5A; Ap5A and MnCl2; Ap4A; ATP; and ATP.NaF.MgCl2. The aggregation pattern of molecules in the first four crystals is similar to that found in a majority of MsMutT1-NTP crystals. Substrate molecules occupy the primary binding site and ATP occupies a site at an intermolecular interface, in the first two. ATP occupies both the sites in the third and fourth crystal. The protein-ligand interactions observed in these crystal structures lead to an explanation of the molecular mechanism of hydrolysis of ApnA by MsMutT1. The fifth crystal exhibits a new packing arrangement. The structure of the complex provides an explanation for the fluoride inhibition of the activity of the enzyme. It would thus appear that MutT1 has a major role involving the hydrolysis of diadenosine polyphosphates, which could be elucidated at the molecular level.

Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates.

Canals are supramolecular complexes observed in the cell wall of Candida maltosa grown in the presence of hexadecane as a sole carbon source. Such structures were not observed in glucose-grown cells. Microscopic observations of cells stained with diaminobenzidine revealed the presence of oxidative enzymes in the canals. 4΄,6΄-diamino-2-phenylindole staining revealed that a substantial part of cellular polyphosphate was present in the cell wall of cells grown on hexadecane in condition of phosphate limitation. The content and chain length of polyphosphates were higher in hexadecane-grown cells than in glucose grown ones. The treatment of cells with yeast polyphosphatase PPX1 resulted in the decrease of the canal size. These data clearly indicated that polyphosphates are constituents of canals; they might play an important role in the canal structure and functioning.

Ppn2, a novel Zn2+-dependent polyphosphatase in the acidocalcisome-like yeast vacuole.

Acidocalcisome-like organelles are found in all kingdoms of life. Many of their functions, such as the accumulation and storage of metal ions, nitrogen and phosphate, the activation of blood clotting and inflammation, depend on the controlled synthesis and turnover of polyphosphate (polyP), a polymer of inorganic phosphate linked by phosphoric anhydride bonds. The exploration of the role of acidocalcisomes in metabolism and physiology requires the manipulation of polyP turnover, yet the complete set of proteins responsible for this turnover is unknown. Here, we identify a novel type of polyphosphatase operating in the acidocalcisome-like vacuoles of the yeast Saccharomyces cerevisiae, which we called Ppn2. Ppn2 belongs to the PPP-superfamily of metallophosphatases, is activated by Zn2+ ions and exclusively shows endopolyphosphatase activity. It is sorted to vacuoles via the multivesicular body pathway. Together with Ppn1, Ppn2 is responsible for a substantial fraction of polyphosphatase activity that is necessary to mobilize polyP stores, for example in response to phosphate scarcity. This finding opens the way to manipulating polyP metabolism more profoundly and deciphering its roles in phosphate and energy homeostasis, as well as in signaling.

Effects of protonation on the hydrolysis of triphosphate in vacuum and the implications for catalysis by nucleotide hydrolyzing enzymes.

BACKGROUND: Nucleoside triphosphate (NTP) hydrolysis is a key reaction in biology. It involves breaking two very stable bonds (one P-O bond and one O-H bond of water), in either a concurrent or a sequential way. Here, we systematically examine how protonation of the triphosphate affects the mechanism of hydrolysis. RESULTS: The hydrolysis reaction of methyl triphosphate in vacuum is computed with protons in various numbers and position on the three phosphate groups. Protonation is seen to have a strong catalytic effect, with the reaction mechanism depending highly on the protonation pattern. CONCLUSION: This dependence is apparently complicated, but is shown to obey a well-defined set of rules: Protonation of the α- and ß-phosphate groups favors a sequential hydrolysis mechanism, whereas γ-protonation favors a concurrent mechanism, the two effects competing with each other in cases of simultaneous protonation. The rate-limiting step is always the breakup of the water molecule while it attacks the γ-phosphorus, and its barrier is lowered by γ-protonation. This step has significantly lower barriers in the sequential reactions, because the dissociated γ-metaphosphate intermediate (PγO3-) is a much better target for water attack than the un-dissociated γ-phosphate (-PγO42-). The simple chemical logic behind these rules helps to better understand the catalytic strategy used by NTPase enzymes, as illustrated here for the catalytic pocket of myosin. A set of rules was determined that describes how protonating the phosphate groups affects the hydrolysis mechanism of methyl triphosphate: Protonation of the α- and/or ß- phosphate groups promotes a sequential mechanism in which P-O bond breaking precedes the breakup of the attacking water, whereas protonation of the γ-phosphate promotes a concurrent mechanism and lowers the rate-limiting barrier of water breakup. The role played by individual protein residues in the catalytic pocket of triphosphate hydrolysing enzymes can be assigned accordingly.

Phosphorothioate analogs of P1,P3-di(nucleosid-5'-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase.

Di(nucleosid-5'-yl) polyphosphates (NPnN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g., HIT proteins). Their structural analogs may serve as molecular probes and potential therapeutic agents. Three P1,P3-bis-thio-analogs of symmetrical di(nucleosid-5'-yl) triphosphates (NP3N) bearing adenosine, guanosine or ribavirin residues (6, 7 and 8, respectively), were obtained by direct condensation of corresponding base-protected nucleoside-5'-O-(2-thio-1,3,2-oxathiaphospholane) with anhydrous phosphoric acid in the presence of DBU. Deprotected products 6 and 8 were separated into individual P-diastereoisomers, whereas 7 was partially separated to yield diastereomerically enriched fractions. The absolute configuration at P-stereogenic centers in the separated diastereoisomers was assigned by RP-HPLC analysis of the products of enzymatic digestion with snake venom phosphodiesterase. The Fhit-assisted hydrolysis rates for 6 and 7 are by 2-3 orders of magnitude lower than that for the reference AP3A, and depend on the configuration of the stereogenic phosphorus atoms, while 8 occurred to be resistant to this cleavage.

Putative binding mode of Escherichia coli exopolyphosphatase and polyphosphates based on a hybrid in silico/biochemical approach.

The exopolyphosphatase of Escherichia coli processively and completely hydrolyses long polyphosphate chains to ortho-phosphate. Genetic surveys, based on the analysis of single ppx(-) or ppk(-) mutants and on the double mutant, demonstrate a relationship between these genes and the survival capacity. The exopolyphosphatase belongs to the ASKHA protein superfamily, hence, its active site is well known; however, the knowledge of the way in which this enzyme binds polyP remains incomplete. Here we present different computational approaches, site-direct mutagenesis and kinetic data to understand the relationship between structure and function of exopolyphosphatase. We propose H(378) as a fundamental gatekeeper for the recognition of long chain polyphosphate.