Ion-combination specific effects driving the enzymatic activity of halophilic alcohol dehydrogenase 2 from Haloferax volcanii in aqueous ionic liquid solvent mixtures.

Biocatalysis in ionic liquids enables novel routes for bioprocessing. Enzymes derived from extremophiles promise greater stability and activity under ionic liquid (IL) influence. Here, we probe the enzyme alcohol dehydrogenase 2 from the halophilic archaeon Haloferax volcanii in thirteen different ion combinations for relative activity and analyse the results against molecular dynamics (MD) simulations of the same IL systems. We probe the ionic liquid property space based on ion polarizability and molecular electrostatic potential. Using the radial distribution functions, survival probabilities and spatial distribution functions of ions, we show that cooperative ion-ion interactions determine ion-protein interactions, and specifically, strong ion-ion interactions equate to higher enzymatic activity if neither of the ions interact strongly with the protein surface. We further demonstrate a tendency for cations interacting with the protein surface to be least detrimental to enzymatic activity if they show a low polarizability when combined with small hydrophilic anions. We also find that the IL ion influence is not mitigated by the surplus of negatively charged residues of the halophilic enzyme. This is shown by free energy landscape analysis in root mean square deviation and distance variation plots of active site gating residues (Trp43 and His273) demonstrating no protection of specific structural elements relevant to preserving enzymatic activity. On the other hand, we observe a general effect across all IL systems that a tight binding of water at acidic residues is preferentially interrupted at these residues through the increased presence of potassium ions. Overall, this study demonstrates a co-ion interaction dependent influence on allosteric surface residues controlling the active/inactive conformation of halophilic alcohol dehydrogenase 2 and the necessity to engineer ionic liquid systems for enzymes that rely on the integrity of functional surface residues regardless of their halophilicity or thermophilicity for use in bioprocessing.

Exploring the potential application of alternative nuclei in NMR based metabolomics.

INTRODUCTION: Nuclear magnetic resonance (NMR) is widely used in metabolomics but it focusses on 1H over other NMR-active nuclei. OBJECTIVES: To evaluate the potential of alternative NMR-sensitive nuclei to generate useful metabolomic data. METHOD: Proton, carbon, phosphorus, and nitrogen-based NMR metabolomics was undertaken on extracts from mint and European honey bee tissue. RESULTS: Carbon NMR provided useful information but required larger sample sizes. Phosphorus produced overlapping peaks in one dimensional (1D) analysis but showed potential in 2D experiments. 15N NMR was found to not be sensitive enough for general metabolomic work. CONCLUSIONS: Alternative NMR active nuclei are useful for metabolomics.

Over the rainbow: structural characterization of the chromoproteins gfasPurple, amilCP, spisPink and eforRed.

Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp2-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp3-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.

Sensitivity of the stripe-faced dunnart, Sminthopsis macroura (Gould 1845), to the insecticide, fipronil; implications for pesticide risk assessments in Australia.

A lack of toxicity data quantifying responses of Australian native mammals to agricultural pesticides prompted an investigation into the sensitivity of the stripe-faced dunnart, Sminthopsis macroura (Gould 1845) to the insecticide, fipronil (5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinyl pyrazole, CAS No. 120068-37-3). Using the Up-And-Down method for determining acute oral toxicity in mammals (OECD) median lethal dose estimates of 990 mg kg-1 (95% confidence interval (CI) = 580.7-4770.0 mg kg-1) and 270.4 mg kg-1 (95% CI = 0.0->20,000.0 mg kg-1) were resolved for male and female S. macroura, respectively. The difference between median lethal dose estimates for males and females may have been influenced by the older ages of two female dunnarts. Consequently, further modelling of female responses to fipronil doses used the following assumptions: (a) death at 2000 mg kg-1, (b) survival at 500 mg kg-1 and (c) a differential response (both survival and death) at 990 mg kg-1. This modelling revealed median lethal dose estimates for female S. macroura of 669.1 mg kg-1 (95% CI = 550-990 mg kg-1; assuming death at 990 mg kg-1) and 990 mg kg-1 (95% CI = 544.7-1470 mg kg-1; assuming survival at 990 mg kg-1). These median lethal dose estimates are 3-10-fold higher than available LD50 values of 94 mg kg-1 for a similarly sized eutherian mammal, Mus musculus (L. 1758) and 97 mg kg-1 for Rattus norvegicus (Birkenhout 1769). Implications for pesticide risk assessments in Australia are discussed.

Towards a Systems Biology Approach to Understanding the Lichen Symbiosis: Opportunities and Challenges of Implementing Network Modelling.

Lichen associations, a classic model for successful and sustainable interactions between micro-organisms, have been studied for many years. However, there are significant gaps in our understanding about how the lichen symbiosis operates at the molecular level. This review addresses opportunities for expanding current knowledge on signalling and metabolic interplays in the lichen symbiosis using the tools and approaches of systems biology, particularly network modelling. The largely unexplored nature of symbiont recognition and metabolic interdependency in lichens could benefit from applying a holistic approach to understand underlying molecular mechanisms and processes. Together with 'omics' approaches, the application of signalling and metabolic network modelling could provide predictive means to gain insights into lichen signalling and metabolic pathways. First, we review the major signalling and recognition modalities in the lichen symbioses studied to date, and then describe how modelling signalling networks could enhance our understanding of symbiont recognition, particularly leveraging omics techniques. Next, we highlight the current state of knowledge on lichen metabolism. We also discuss metabolic network modelling as a tool to simulate flux distribution in lichen metabolic pathways and to analyse the co-dependence between symbionts. This is especially important given the growing number of lichen genomes now available and improved computational tools for reconstructing such models. We highlight the benefits and possible bottlenecks for implementing different types of network models as applied to the study of lichens.

A method for topical dosing of invertebrates with pesticide for use in feeding experiments.

The ability to produce large numbers of pesticide-exposed insects (e.g. crickets) is important for feeding studies into the effects of pesticides on key predatory species. House crickets (Acheta domesticus L. 1758) were submersed in serial dilutions of the pesticides, fenitrothion and fipronil, used for the control of locusts in Australia, and then rapidly frozen for residue analysis. Good correlations were found between increasing concentrations of serial pesticide dilutions and the resultant residual concentrations of the parent compounds in crickets, with R2 values of 0.949 (fenitrothion) and 0.946 (fipronil). R2 values for the much less abundant fipronil metabolites were lower 0.858 (sulfone), 0.368 (desulfinyl) and 0.785 (sulfide). This method enables insecticide exposure mimicking the field conditions to be assessed, and can be done immediately prior to an experiment. This ensures locusts remain alive when introduced to the feeding chambers, and enables multiple prey items to be dosed with a known pesticide burden.

Plant expression of NifD protein variants resistant to mitochondrial degradation.

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F420-0 in Mycobacteria.

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

Bacterial catabolism of s-triazine herbicides: biochemistry, evolution and application.

The synthetic s-triazines are abundant, nitrogen-rich, heteroaromatic compounds used in a multitude of applications including, herbicides, plastics and polymers, and explosives. Their presence in the environment has led to the evolution of bacterial catabolic pathways in bacteria that allow use of these anthropogenic chemicals as a nitrogen source that supports growth. Herbicidal s-triazines have been used since the mid-twentieth century and are among the most heavily used herbicides in the world, despite being withdrawn from use in some areas due to concern about their safety and environmental impact. Bacterial catabolism of the herbicidal s-triazines has been studied extensively. Pseudomonas sp. strain ADP, which was isolated more than thirty years after the introduction of the s-triazine herbicides, has been the model system for most of these studies; however, several alternative catabolic pathways have also been identified. Over the last five years, considerable detail about the molecular mode of action of the s-triazine catabolic enzymes has been uncovered through acquisition of their atomic structures. These structural studies have also revealed insights into the evolutionary origins of this newly acquired metabolic capability. In addition, s-triazine-catabolizing bacteria and enzymes have been used in a range of applications, including bioremediation of herbicides and cyanuric acid, introducing metabolic resistance to plants, and as a novel selectable marker in fermentation organisms. In this review, we cover the discovery and characterization of bacterial strains, metabolic pathways and enzymes that catabolize the s-triazines. We also consider the evolution of these new enzymes and pathways and discuss the practical applications that have been considered for these bacteria and enzymes. One Sentence Summary: A detailed understanding of bacterial herbicide catabolic enzymes and pathways offer new evolutionary insights and novel applied tools.

A Miniature Gas Sampling Interface with Open Microfluidic Channels: Characterization of Gas-to-Liquid Extraction Efficiency of Volatile Organic Compounds.

Chemosensory protein based olfactory biosensors are expected to play a significant role in next-generation volatile organic compound (VOC) detection systems due to their ultra-high sensitivity and selectivity. As these biosensors can perform most efficiently in aqueous environments, the detection systems need to incorporate a gas sampling interface for gas-to-liquid extraction. This interface should extract the VOCs from the gas phase with high efficiency and transfer them into the liquid containing biosensors to enable subsequent detection. To design such a transfer interface, an understanding of the key parameters influencing the gas-to-liquid extraction efficiency of target VOCs is crucial. This paper reports a gas sampling interface system based on a microfluidic open-channel device for gas-to-liquid extraction. By using this device as a model platform, the key parameters dictating the VOC extraction efficiency were identified. When loaded with 30 µL of capture liquid, the microfluidic device generates a gas-liquid interface area of 3 cm2 without using an interfacial membrane. The pumpless operation based on capillary flow was demonstrated for capture liquid loading and collection. Gas samples spiked with lipophilic model volatiles (hexanal and allyl methyl sulfide) were used for characterization of the VOC extraction efficiency. Decreasing the sampling temperature to 15 °C had a significant impact on increasing capture efficiency, while variation in the gas sampling flow rate had no significant impact in the range between 40-120 mL min-1. This study found more than a 10-fold increase in capture efficiency by chemical modification of the capture liquid with alpha-cyclodextrin. The highest capture efficiency of 30% was demonstrated with gas samples spiked with hexanal to a concentration of 16 ppm (molar proportion). The approach in this study should be useful for further optimisation of miniaturised gas-to-liquid extraction systems and contribute to the design of chemosensory protein-based VOC detection systems.

Computer-Guided Surface Engineering for Enzyme Improvement.

Protein engineering strategies are often guided by our understanding of how the structure of a protein determines its function. However, our understanding is generally restricted to small regions of a protein, namely the active site and its immediate vicinity, while the remainder of the protein is something of an enigma. Studying highly homologous transaminases with strictly conserved active sites, but different substrate preferences and activities, we predict and experimentally validate that the surface of the protein far from the active site carries out a decisive role in substrate selectivity and catalytic efficiency. Using a unique molecular dynamics approach and novel trajectory analysis, we demonstrate the phenomenon of surface-directed ligand diffusion in this well-known protein family for the first time. Further, we identify the residues involved in directing substrate, design surface channel variants endowed for improved kinetic properties and establish a broadly applicable new approach for protein engineering.

Decoding the Rich Biological Properties of Noble Gases: How Well Can We Predict Noble Gas Binding to Diverse Proteins?

The chemically inert noble gases display a surprisingly rich spectrum of useful biological properties. Relatively little is known about the molecular mechanisms behind these effects. It is clearly not feasible to conduct large numbers of pharmacological experiments on noble gases to identify activity. Computational studies of the binding of noble gases and proteins can address this paucity of information and provide insight into mechanisms of action. We used bespoke computational grid calculations to predict the positions of energy minima in the interactions of noble gases with diverse proteins. The method was validated by quantifying how well simulations could predict binding positions in 131 diverse protein X-ray structures containing 399 Xe and Kr atoms. We found excellent agreement between calculated and experimental binding positions of noble gases. 94 % of all crystallographic xenon atoms were within 1 Xe van der Waals (vdW) diameter of a predicted binding site, and 97 % lay within 2 vdW diameters. 100 % of crystallographic krypton atoms were within 1 Kr vdW diameter of a predicted binding site. We showed the feasibility of large-scale computational screening of all ≈60 000 unique structures in the Protein Data Bank. This will elucidate biochemical mechanisms by which these novel 'atomic drugs' elicit their valuable biochemical properties and identify new medical uses.

Isolation of the (+)-Pinoresinol-Mineralizing Pseudomonas sp. Strain SG-MS2 and Elucidation of Its Catabolic Pathway.

Pinoresinol is a dimer of two ß-ß'-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several ß-ß-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2.IMPORTANCE The oxidative catabolism of (+)-pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.

Cofactor Tail Length Modulates Catalysis of Bacterial F420-Dependent Oxidoreductases.

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.

Design of silk proteins with increased heme binding capacity and fabrication of silk-heme materials.

In our previous studies, heme was bound into honeybee silk to generate materials that could function as nitric oxide sensors or as recoverable heterogeneous biocatalysts. In this study, we sought to increase the heme-binding capacity of the silk protein by firstly redesigning the heme binding site to contain histidine as the coordinating residue and secondly, by adding multiple histidine residues within the core of the coiled coil core region of the modified silk protein. We used detergent and a protein denaturant to confirm the importance of the helical structure of the silk for heme coordination. Aqueous methanol treatment, which was used to stabilize the materials, transformed the low-spin, six-coordinate heme to a five-coordinate high-spin complex, thus providing a vacant site for ligand binding. The optimal aqueous methanol treatment time that simultaneously maintains the helical protein structure and stabilizes the silk material without substantial leaching of heme from the system was determined.

A Motif in the F Homomorph of Rabbit Haemorrhagic Disease Virus Polymerase Is Important for the Subcellular Localisation of the Protein and Its Ability to Induce Redistribution of Golgi Membranes.

Rabbit haemorrhagic disease virus (RHDV) is a calicivirus that infects and frequently kills rabbits. Previously, we showed that the RHDV RNA-dependent RNA polymerase (RdRp) is associated with distinct, but yet uncharacterised subcellular structures and is capable of inducing a redistribution of Golgi membranes. In this study, we identified a partially hidden hydrophobic motif that determines the subcellular localisation of recombinant RHDV RdRp in transfected cells. This novel motif, 189LLWGCDVGVAVCAAAVFHNICY210, is located within the F homomorph, between the conserved F3 and A motifs of the core RdRp domain. Amino acid substitutions that decrease the hydrophobicity of this motif reduced the ability of the protein to accumulate in multiple subcellular foci and to induce a rearrangement of the Golgi network. Furthermore, preliminary molecular dynamics simulations suggest that the RHDV RdRp could align with the negatively charged surfaces of biological membranes and undergo a conformational change involving the F homomorph. These changes would expose the newly identified hydrophobic motif so it could immerse itself into the outer leaflet of intracellular membranes.

Comparative Lipidomics and Proteomics of Lipid Droplets in the Mesocarp and Seed Tissues of Chinese Tallow (Triadica sebifera).

Lipid droplets (LDs) are composed of a monolayer of phospholipids (PLs), surrounding a core of non-polar lipids that consist mostly of triacylglycerols (TAGs) and to a lesser extent diacylglycerols. In this study, lipidome analysis illustrated striking differences in non-polar lipids and PL species between LDs derived from Triadica sebifera seed kernels and mesocarp. In mesocarp LDs, the most abundant species of TAG contained one C18:1 and two C16:0 and fatty acids, while TAGs containing three C18 fatty acids with higher level of unsaturation were dominant in the seed kernel LDs. This reflects the distinct differences in fatty acid composition of mesocarp (palmitate-rich) and seed-derived oil (α-linoleneate-rich) in T. sebifera. Major PLs in seed LDs were found to be rich in polyunsaturated fatty acids, in contrast to those with relatively shorter carbon chain and lower level of unsaturation in mesocarp LDs. The LD proteome analysis in T. sebifera identified 207 proteins from mesocarp, and 54 proteins from seed kernel, which belong to various functional classes including lipid metabolism, transcription and translation, trafficking and transport, cytoskeleton, chaperones, and signal transduction. Oleosin and lipid droplets associated proteins (LDAP) were found to be the predominant proteins associated with LDs in seed and mesocarp tissues, respectively. We also show that LDs appear to be in close proximity to a number of organelles including the endoplasmic reticulum, mitochondria, peroxisomes, and Golgi apparatus. This comparative study between seed and mesocarp LDs may shed some light on the structure of plant LDs and improve our understanding of their functionality and cellular metabolic networks in oleaginous plant tissues.

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism.

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,ß-unsaturated compounds.

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.

F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.