Bioprospecting of Novel Extremozymes From Prokaryotes-The Advent of Culture-Independent Methods.

Extremophiles are remarkable organisms that thrive in the harshest environments on Earth, such as hydrothermal vents, hypersaline lakes and pools, alkaline soda lakes, deserts, cold oceans, and volcanic areas. These organisms have developed several strategies to overcome environmental stress and nutrient limitations. Thus, they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance. Genetic and structural information derived from extremophiles and extremozymes can be used for bioengineering other nontolerant enzymes. Furthermore, extremophiles can be a valuable resource for novel biotechnological and biomedical products due to their biosynthetic properties. However, understanding life under extreme conditions is challenging due to the difficulties of in vitro cultivation and observation since > 99% of organisms cannot be cultivated. Consequently, only a minor percentage of the potential extremophiles on Earth have been discovered and characterized. Herein, we present a review of culture-independent methods, sequence-based metagenomics (SBM), and single amplified genomes (SAGs) for studying enzymes from extremophiles, with a focus on prokaryotic (archaea and bacteria) microorganisms. Additionally, we provide a comprehensive list of extremozymes discovered via metagenomics and SAGs.

Understanding High-Salt and Cold Adaptation of a Polyextremophilic Enzyme.

The haloarchaeon Halorubrum lacusprofundi is among the few polyextremophilic organisms capable of surviving in one of the most extreme aquatic environments on Earth, the Deep Lake of Antarctica (-18 °C to +11.5 °C and 21-28%, w/v salt content). Hence, H. lacusprofundi has been proposed as a model for biotechnology and astrobiology to investigate potential life beyond Earth. To understand the mechanisms that allow proteins to adapt to both salinity and cold, we structurally (including X-ray crystallography and molecular dynamics simulations) and functionally characterized the ß-galactosidase from H. lacusprofundi (hla_bga). Recombinant hla_bga (produced in Haloferax volcanii) revealed exceptional stability, tolerating up to 4 M NaCl and up to 20% (v/v) of organic solvents. Despite being cold-adapted, hla_bga was also stable up to 60 °C. Structural analysis showed that hla_bga combined increased surface acidity (associated with halophily) with increased structural flexibility, fine-tuned on a residue level, for sustaining activity at low temperatures. The resulting blend enhanced structural flexibility at low temperatures but also limited protein movements at higher temperatures relative to mesophilic homologs. Collectively, these observations help in understanding the molecular basis of a dual psychrophilic and halophilic adaptation and suggest that such enzymes may be intrinsically stable and functional over an exceptionally large temperature range.

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase.

Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon Halobacterium sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme's salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution.

Arabidopsis DIACYLGLYCEROL KINASE4 is involved in nitric oxide-dependent pollen tube guidance and fertilization.

Nitric oxide (NO) is a key signaling molecule that regulates diverse biological processes in both animals and plants, including important roles in male gamete physiology. In plants, NO is generated in pollen tubes (PTs) and affects intracellular responses through the modulation of Ca2+ signaling, actin organization, vesicle trafficking and cell wall deposition, bearing consequences in pollen-stigma interactions and PT guidance. In contrast, the NO-responsive proteins that mediate these responses remain elusive. Here, we show that PTs of Arabidopsis thaliana mutants impaired in the pollen-specific DIACYLGLYCEROL KINASE4 (DGK4) grow slower and become partially insensitive to NO-dependent growth inhibition and re-orientation responses. Recombinant DGK4 protein yields NO-responsive spectral and catalytic changes in vitro that are compatible with a role in NO perception and signaling in PTs. In addition to the expected phosphatidic acid-producing kinase activity, DGK4 recombinant protein also revealed guanylyl cyclase activity, as inferred by sequence analysis. Our results are compatible with a role for the fast-diffusible NO gas in signaling and cell-cell communication via the modulation of DGK4 activity during the progamic phase of angiosperm reproduction.

Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNAPyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.

Genetically Encoded Biotin Analogues: Incorporation and Application in Bacterial and Mammalian Cells.

The biotin-streptavidin interaction is among the strongest known in nature. Herein, the site-directed incorporation of biotin and 2-iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. By using the high-affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well-defined protein-drug conjugates.

A polyextremophilic alcohol dehydrogenase from the Atlantis II Deep Red Sea brine pool.

Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl-methyl-ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo- and thermoadaptation.

Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions.

Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS).

Bedford-Type Palladacycle-Catalyzed Miyaura Borylation of Aryl Halides with Tetrahydroxydiboron in Water.

A mild aqueous protocol for palladium catalyzed Miyaura borylation of aryl iodides, aryl bromides and aryl chlorides with tetrahydroxydiboron (BBA) as a borylating agent is developed. The developed methodology requires low catalyst loading of Bedford-type palladacycle catalyst (0.05 mol %) and works best under mild reaction conditions at 40 °C in short time of 6 h in water. In addition, our studies show that for Miyaura borylation using BBA in aqueous condition, maintaining a neutral reaction pH is very important for reproducibility and higher yields of corresponding borylated products. Moreover, our protocol is applicable for a broad range of aryl halides, corresponding borylated products are obtained in excellent yields up to 93% with 29 examples demonstrating its broad utility and functional group tolerance.

Identification and Experimental Characterization of an Extremophilic Brine Pool Alcohol Dehydrogenase from Single Amplified Genomes.

Because only 0.01% of prokaryotic genospecies can be cultured and in situ observations are often impracticable, culture-independent methods are required to understand microbial life and harness potential applications of microbes. Here, we report a methodology for the production of proteins with desired functions based on single amplified genomes (SAGs) from unculturable species. We use this method to resurrect an alcohol dehydrogenase (ADH/D1) from an uncharacterized halo-thermophilic archaeon collected from a brine pool at the bottom of the Red Sea. Our crystal structure of 5,6-dihydroxy NADPH-bound ADH/D1 combined with biochemical analyses reveal the molecular features of its halo-thermophily, its unique habitat adaptations, and its possible reaction mechanism for atypical oxygen activation. Our strategy offers a general guide for using SAGs as a source for scientific and industrial investigations of "microbial dark matter."

cis-Tetrachlorido-bis(indazole)osmium(iv) and its osmium(iii) analogues: paving the way towards the cis-isomer of the ruthenium anticancer drugs KP1019 and/or NKP1339.

The relationship between cis-trans isomerism and anticancer activity has been mainly addressed for square-planar metal complexes, in particular, for platinum(ii), e.g., cis- and trans-[PtCl2(NH3)2], and a number of related compounds, of which, however, only cis-counterparts are in clinical use today. For octahedral metal complexes, this effect of geometrical isomerism on anticancer activity has not been investigated systematically, mainly because the relevant isomers are still unavailable. An example of such an octahedral complex is trans-[RuCl4(Hind)2]-, which is in clinical trials now as its indazolium (KP1019) or sodium salt (NKP1339), but the corresponding cis-isomers remain inaccessible. We report the synthesis of Na[cis-OsIIICl4(κN2-1H-ind)2]·(Na[1]) suggesting a route to the cis-isomer of NKP1339. The procedure involves heating (H2ind)[OsIVCl5(κN1-2H-ind)] in a high boiling point organic solvent resulting in an Anderson rearrangement with the formation of cis-[OsIVCl4(κN2-1H-ind)2] ([1]) in high yield. The transformation is accompanied by an indazole coordination mode switch from κN1 to κN2 and stabilization of the 1H-indazole tautomer. Fully reversible spectroelectrochemical reduction of [1] in acetonitrile at 0.46 V vs. NHE is accompanied by a change in electronic absorption bands indicating the formation of cis-[OsIIICl4(κN2-1H-ind)2]- ([1]-). Chemical reduction of [1] in methanol with NaBH4 followed by addition of nBu4NCl afforded the osmium(iii) complex nBu4N[cis-OsIIICl4(κN2-1H-ind)2] (nBu4N[1]). A metathesis reaction of nBu4N[1] with an ion exchange resin led to the isolation of the water-soluble salt Na[1]. The X-ray diffraction crystal structure of [1]·Me2CO was determined and compared with that of trans-[OsIVCl4(κN2-1H-ind)2]·2Me2SO (2·2Me2SO), also prepared in this work. EPR spectroscopy was performed on the OsIII complexes and the results were analyzed by ligand-field and quantum chemical theories. We furthermore assayed effects of [1] and Na[1] on cell viability and proliferation in comparison with trans-[OsIVCl4(κN1-2H-ind)2] [3] and cisplatin and found a strong reduction of cell viability at concentrations between 30 and 300 µM in different cancer cell lines (HT29, H446, 4T1 and HEK293). HT-29 cells are less sensitive to cisplatin than 4T1 cells, but more sensitive to [1] and Na[1], as shown by decreased proliferation and viability as well as an increased late apoptotic/necrotic cell population.

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting.

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery.

The delivery of large cargos of diameter above 15nm for biomedical applications has proved challenging since it requires biocompatible, stably-loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica-iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60nm in diameter and post-functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP-Ferritin, ~534kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica-iron oxide nanovectors, which is thus a promising for biomedical applications.

Near-Infrared Intraoperative Chemiluminescence Imaging.

Intraoperative imaging technologies recently entered the operating room, and their implementation is revolutionizing how physicians plan, monitor, and perform surgical interventions. In this work, we present a novel surgical imaging reporter system: intraoperative chemiluminescence imaging (ICI). To this end, we have leveraged the ability of a chemiluminescent metal complex to generate near-infrared light upon exposure to an aqueous solution of Ce(4+) in the presence of reducing tissue or blood components. An optical camera spatially resolves the resulting photon flux. We describe the construction and application of a prototype imaging setup, which achieves a detection limit as low as 6.9 pmol cm(-2) of the transition-metal-based ICI agent. As a proof of concept, we use ICI for the in vivo detection of our transition metal tracer following both systemic and subdermal injections. The very high signal-to-noise ratios make ICI an interesting candidate for the development of new intraoperative imaging technologies.

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.

Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe4S4] Enzyme IspG (GcpE).

IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent and an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG-catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate into (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate. In addition, the enzyme's structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism, which describes all stages from initial ring opening to formation of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many prokaryotic and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.

Mechanistic insights into the reductive dehydroxylation pathway for the biosynthesis of isoprenoids promoted by the IspH enzyme.

Here, we report an integrated quantum mechanics/molecular mechanics (QM/MM) study of the bio-organometallic reaction pathway of the 2H+/2e- reduction of (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into the so called universal terpenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), promoted by the IspH enzyme. Our results support the viability of the bio-organometallic pathway through rotation of the OH group of HMBPP away from the [Fe4S4] cluster at the core of the catalytic site, to become engaged in a H-bond with Glu126. This rotation is synchronous with π-coordination of the C2[double bond, length as m-dash]C3 double bond of HMBPP to the apical Fe atom of the [Fe4S4] cluster. Dehydroxylation of HMBPP is triggered by a proton transfer from Glu126 to the OH group of HMBPP. The reaction pathway is completed by competitive proton transfer from the terminal phosphate group to the C2 or C4 atom of HMBPP.

Mining a database of single amplified genomes from Red Sea brine pool extremophiles-improving reliability of gene function prediction using a profile and pattern matching algorithm (PPMA).

Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Intrinsic sequencing data quality problems of single amplified genomes (SAGs) and poor homology of novel extremophile's genomes pose significant challenges for the attribution of functions to the coding sequences identified. The anoxic deep-sea brine pools of the Red Sea are a promising source of novel enzymes with unique evolutionary adaptation. Sequencing data from Red Sea brine pool cultures and SAGs are annotated and stored in the Integrated Data Warehouse of Microbial Genomes (INDIGO) data warehouse. Low sequence homology of annotated genes (no similarity for 35% of these genes) may translate into false positives when searching for specific functions. The Profile and Pattern Matching (PPM) strategy described here was developed to eliminate false positive annotations of enzyme function before progressing to labor-intensive hyper-saline gene expression and characterization. It utilizes InterPro-derived Gene Ontology (GO)-terms (which represent enzyme function profiles) and annotated relevant PROSITE IDs (which are linked to an amino acid consensus pattern). The PPM algorithm was tested on 15 protein families, which were selected based on scientific and commercial potential. An initial list of 2577 enzyme commission (E.C.) numbers was translated into 171 GO-terms and 49 consensus patterns. A subset of INDIGO-sequences consisting of 58 SAGs from six different taxons of bacteria and archaea were selected from six different brine pool environments. Those SAGs code for 74,516 genes, which were independently scanned for the GO-terms (profile filter) and PROSITE IDs (pattern filter). Following stringent reliability filtering, the non-redundant hits (106 profile hits and 147 pattern hits) are classified as reliable, if at least two relevant descriptors (GO-terms and/or consensus patterns) are present. Scripts for annotation, as well as for the PPM algorithm, are available through the INDIGO website.

A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.

Phenylalanine--a biogenic ligand with flexible η6- and η6:κ1-coordination at ruthenium(II) centres.

The reaction of (S)-2,5-dihydrophenylalanine 1 with ruthenium(III) chloride yields the µ-chloro-bridged dimeric η(6)-phenylalanine ethyl ester complex 3, which can be converted into the monomeric analogue, η(6):κ(1)-phenylalanine ethyl ester complex 12, under basic conditions. Studies were carried out to determine the stability and reactivity of complexes bearing η(6)- and η(6):κ(1)-chelating phenylalanine ligands under various conditions. Reaction of 3 with ethylenediamine derivatives N-p-tosylethylenediamine or 1,4-di-N-p-tosylethylenediamine results in the formation of monomeric η(6):κ(1)-phenylalanine ethyl ester complexes 14 and 15, which could be saponified yielding complexes 16 and 17 without changing the inner coordination sphere of the metal centre. The structure of η(6):κ(1)-phenylalanine complex 17 and an N-κ(1)-phenylalanine complex 13 resulting from the reaction of 3 with an excess of pyridine were confirmed by X-ray crystallography.