Hyperthermophilic Aquifex Aeolicus Research Articles

Prediction of stability changes upon mutation in an icosahedral capsid.

ABSTRACTIdentifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid. Proteins 2015; 83:1733–1741. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc

Structural membrane proteomics focused on respiratory protein complexes of hyperthermophilic eubacterium Aquifex aeolicus

Structural membrane proteomics focused on respiratory protein complexes of hyperthermophilic eubacterium Aquifex aeolicus

Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production

How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein-protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.

Dynamics measured by neutron scattering correlates with the organization of bioenergetics complexes in natural membranes from hyperthermophile and mesophile bacteria

Various models on membrane structure and organization of proteins and complexes in natural membranes emerged during the last years. However, the lack of systematic dynamical studies to complement structural investigations hindered the establishment of a more complete picture of these systems. Elastic incoherent neutron scattering gives access to the dynamics on a molecular level and was applied to natural membranes extracted from the hyperthermophile Aquifex aeolicus and the mesophile Wolinella succinogenes bacteria. The results permitted to extract a hierarchy of dynamic flexibility and atomic resilience within the samples, which correlated with the organization of proteins in bioenergetics complexes and the functionality of the membranes.

Fabrication of uniform layer-by-layer assemblies with complementary protein cage nanobuilding blocks via simple His-tag/metal recognition.

A capsid-forming enzyme, lumazine synthase isolated from hyperthermophile Aquifex aeolicus (AaLS), is prepared and utilized as a template for constructing nanobuilding blocks to fabricate uniform layer-by-layer (LbL) assemblies. Two functionally complementary AaLS protein cage nanoparticles (PCNs) are generated either by genetically introducing His-tags on the surface of wild-type AaLS PCNs or by chemically attaching metal chelates (Ni-NTA moiety) to the surface of cysteine-bearing AaLS PCNs individually. The multivalent displays of His-tags (AaLS-His6 PCN) and Ni-NTA ligands (AaLS-NTA-Ni PCN) on the surface of each complementary AaLS PCN are successfully demonstrated by mass spectrometric and surface plasmon resonance analyses. By using these two complementary AaLS PCNs, uniform LbL assemblies are constructed via simple recognition between His-tags and metal chelates without the aid of additional binding mediators. This approach illustrates the potential of fabricating uniform nanostructures using protein-based hybrid functional nanobuilding blocks.

The CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold.

The Elusive Third Subunit IIa of the Bacterial B-Type Oxidases: The Enzyme from the Hyperthermophile Aquifex aeolicus

The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba 3-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix “subunit IIa”, which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA2) of 59000 Da, subunit II (encoded by coxB2) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba 3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.

Purification and characterization of the lipid A 4’‐kinase

The chief component of the outer membrane in Gram‐negative bacteria is lipopolysaccharide (LPS). LPS is anchored to the outer leaflet of the outer membrane by the highly immunogenic acylated disaccharide of glucosamine known as lipid A. Since most Gram‐negative bacteria require lipid A for viability, any enzyme responsible for the biosynthesis of this molecule presents itself as an attractive target for the development of novel antimicrobials. In E. coli, the sixth enzyme in the constitutive pathway of lipid A biosynthesis is an integral membrane protein designated LpxK, which phosphorylates the 4′‐position on the distal glucosamine of disaccharide‐1‐phosphate (DSMP) to form lipid IVA. Previous studies of E. coli LpxK have been hampered due to its instability in the presence of detergents, but the ortholog from the hyperthermophile Aquifex aeolicus (AaLpxK) is stable when separated from bacterial membranes and can readily act upon the E. coli‐derived substrate. These properties have allowed for the purification of AaLpxK, the development of a sensitive radioassay for kinase activity, and the screening of crystallization conditions. Activity of purified AaLpxK in vitro has been shown to be dependent upon maintenance of high pH, select divalent cations, and the presence of detergent in the assay system. This research was funded by N.I.H. grant GM51310 to C.R.H. Raetz.

Conferment of Folding Ability to a Naturally Unfolded Apocytochrome c through Introduction of Hydrophobic Amino Acid Residues

Hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic Pseudomonas aeruginosa cytochrome c(551) (PA c(551)), which is structurally homologous to AA c(555) in the holo state. Here we hypothesized that the exceptional apo AA c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a PA c(551) variant (denoted as PA-nh) carrying the nine hydrophobic residues at structurally corresponding positions. Circular dichroism experiments showed that the apo PA-nh variant became folded, unlike the wild-type apo PA c(551), and exhibited much higher stability than the wild type. Another difference between the holo forms of AA c(555) and PA c(551) is the existence of an extra helix in the former. Introduction of the amino acid residues forming the extra helix of AA c(555) into the PA-nh variant did not significantly affect its folding ability in the apo state. Therefore, the nine hydrophobic residues introduced into the apo PA-nh variant were enough to confer the folding ability. PA c(551) represents the first example of the conversion of an intrinsically unfolded apocytochrome c into an autonomously folded one, which was revealed by means of a protein engineering method without heme. Although heme is generally considered to be a trigger of apocytochrome c folding, the present results demonstrate a new heme-independent folding mechanism.

Stabilization Role of a Phenothiazine Derivative on the Electrocatalytic Oxidation of Hydrogen via Aquifex aeolicus Hydrogenase at Graphite Membrane Electrodes

The [NiFe] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic Aquifex aeolicus bacterium (Aa Hase) presents oxygen, carbon monoxide, and temperature resistances. Since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. Efficient immobilization of the enzyme onto electrodes is however a mandatory step. To gain further insight into the parameters governing the interfacial electron process, cyclic voltammetry was performed combining the use of a phenothiazine dye with a membrane electrode design where the enzyme is entrapped in a thin layer. In the absence of the phenothiazine dye, direct electron transfer (DET) for H(2) oxidation is observed due to Aa Hase adsorbed onto the PG electrode. An unexpected loss of the catalytic current with time is however observed. The effect of toluidine blue O (TBO) on the catalytic process is first studied with TBO in solution. In addition to the expected mediated electron transfer process (MET), TBO is demonstrated to reconnect directly some Aa Hase molecules possibly released from the electrode but still entrapped in the thin layer. On adsorbed TBO the two same processes occur demonstrating the ability of the TBO film to connect Aa Hase via a DET process. Loss of activity is however observed due to the poor stability of adsorbed TBO at high temperatures. Aa Hase immobilization is then studied on electropolymerized TBO (pTBO). The effect of film thickness, temperature, presence of inhibitors and pH is evaluated. Given a film thickness less than 20 nm, H(2) oxidation proceeds via a mixed DET/MET process through the pTBO film. A high and very stable H(2) oxidation activity is reached, showing the potential applicability of the bioelectrode for biotechnologies. Finally, the multifunctional roles of TBO-based matrix are underlined, including redox mediator, Aa Hase anchor, but also buffering and ROS scavenger capabilities to drive pH local changes and avoid oxidative damage.

Structural and functional characterization of respiratory supercomplex containing complex III and IV from hyperthermophilic eubacterium Aquifex aeolicus

Structural and functional characterization of respiratory supercomplex containing complex III and IV from hyperthermophilic eubacterium Aquifex aeolicus

Structure of SurE protein fromAquifex aeolicusVF5 at 1.5 Å resolution

SurE is a stationary-phase survival protein found in bacteria, eukaryotes and archaea that exhibits a divalent-metal-ion-dependent phosphatase activity and acts as a nucleotidase and polyphosphate phosphohydrolase. The structure of the SurE protein from the hyperthermophile Aquifex aeolicus has been solved at 1.5 A resolution using molecular replacement with one dimer in the asymmetric unit and refined to an R factor of 15.6%. The crystal packing reveals that two dimers assemble to form a tetramer, although gel-filtration chromatography showed the presence of only a dimer in solution. The phosphatase active-site pocket was occupied by sulfate ions from the crystallization medium.

Heme Is Not Required forAquifex aeolicusCytochromec555Polypeptide Folding

In cytochrome c, it has been supposed that heme must bind to the apo polypeptide for structure formation. We constructed a C12A/C15A variant of hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) in which the covalently heme-binding Cys residues were replaced by Ala, and characterized its molecular features. The apo C12A/C15A variant had almost the same helical content as holo AA c(555), and spontaneously incorporated heme in vitro with no helical content change. These results suggest that the apo AA c(555) polypeptide is intrinsically structured without heme binding, this being the first case of a cytochrome c polypeptide. This finding provides a new suggestion as to cytochrome c formation, that heme is not necessarily required for cytochrome c polypeptide folding.

WaaA of the Hyperthermophilic Bacterium Aquifex aeolicus Is a Monofunctional 3-Deoxy-d-manno-oct-2-ulosonic Acid Transferase Involved in Lipopolysaccharide Biosynthesis

The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.

Dynamics in Thermotoga neapolitana Adenylate Kinase: 15N Relaxation and Hydrogen−Deuterium Exchange Studies of a Hyperthermophilic Enzyme Highly Active at 30 °C

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.

S11.14 Isolation and characterization of cytochrome c oxidase and its supercomplex from the hyperthermophilic eubacterium Aquifex aeolicus

S11.14 Isolation and characterization of cytochrome c oxidase and its supercomplex from the hyperthermophilic eubacterium Aquifex aeolicus

Inhibition of Lipid A Biosynthesis as the Primary Mechanism of CHIR-090 Antibiotic Activity in Escherichia coli

The deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against Pseudomonas aeruginosa and Escherichia coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of E. coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1, and k6 = 0.18 min-1. CHIR-090 at low nanomolar levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including P. aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 microM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 microg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway.

Crystal Structure of Prephenate Dehydrogenase from Aquifex aeolicus: INSIGHTS INTO THE CATALYTIC MECHANISM

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.

Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer

Recent studies have shown that an endogenous lipoperoxidation product, 9-hydroxystearic acid (9-HSA), acts in colon carcinoma cells (HT29) as a growth inhibitor by inducing p21(WAF1) in an immediate-early, p53-independent manner and that p21(WAF1) is required for 9-HSA-mediated growth arrest in HT29 cells. It is conceivable, therefore, to hypothesize that the cytostatic effect induced by this agent is at least partially associated with a molecular mechanism that involves histone deacetylase 1 (HDAC1) inhibition, as demonstrated for sodium butyrate and other specific inhibitors, such as trichostatin A and hydroxamic acids. Here, we show that, after administration, 9-HSA causes an accumulation of hyperacetylated histones and strongly inhibits the activity of HDAC1. The interaction of 9-HSA with the catalytic site of the enzyme has been highlighted by computational modeling of the human HDAC1, using its homolog from the hyperthermophilic Aquifex aeolicus as a template. Consistent with the experimental data, we find that 9-HSA can bind to the active site of the protein, showing that the inhibition of the enzyme can be explained at the molecular level by the ligand-protein interaction.

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold

BackgroundaaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown.ResultsHere we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands.ConclusionWe conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.