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Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na + hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated. Halophilic organisms thrive in high salt conditions and express proteins that display desirable characteristics for industrial applications. Here, the authors use a rational design approach to transform wild-type carbonic anhydrase into a strongly halophilic enzyme.

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 can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in . 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.

F 420 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, F 420 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 F 420 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 F 420 , with oligoglutamate sidechains of different lengths. To understand the evolution of F 420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the F o precursor to F 420 originated in methanogens, F 420 itself was first synthesized in an ancestral actinobacterium. F 420 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.

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in kcat/KM for malathion. X-ray crystal structures for the PTEAr Ser308Leu\\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.

Two classes of F(420)-dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis. One class, the FDR-A enzymes, has up to 100 times more activity than the other. F(420) is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria. We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales, finding that nine can also use F(420)H(2) to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales. The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,β-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F(420) to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products.

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.

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 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 . 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 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.

The antibiotic 2-nitroimidazole (2NI) or azomycin, used for treating drug-resistant tuberculosis and imaging tumor hypoxia, requires activation by bacterial nitroreductases for its antibiotic and cytotoxic effect. Mycobacterium sp. JS330 produces 2-nitroimidazole nitrohydrolase (NnhA) that circumvents 2NI activation, conferring 2NI resistance by hydrolysing it to nitrite and imidazol-2-one (IM2O) instead. This study elucidates NnhA’s structure, catalytic mechanism, and evolutionary background within the guanidino-group modifying enzyme (GME) superfamily, aided by a more soluble protein variant engineered through directed evolution. Despite low sequence similarity and limited occurrence in a few soil-dwelling mycobacteria and Actinomycetota, NnhA maintains the α/β propeller fold characteristic of GME superfamily enzymes and forms an unusual hexameric ring structure formed by a trimer of domain-swapped dimers. The similarity of its active site to arginine deiminases (ADIs) and human dimethylarginine dimethylaminohydrolases (DDAHs), along with molecular dynamics simulations, suggests NnhA’s catalytic mechanism resembles the hydrolysis reactions of these related enzymes. Experimentally determined structure of 2-nitroimidazole hydrolase and its comparison with other related enzymes shed light on the mechanism of action of an unusual antibiotic degrading enzyme in soil bacteria.

The delivery of food security via continued crop yield improvement alone is not an effective food security strategy, and must be supported by pre- and post-border biosecurity policies to guard against perverse outcomes. In the wake of the green revolution, yield gains have been in steady decline, while post-harvest crop losses have increased as a result of insufficiently resourced and uncoordinated efforts to control spoilage throughout global transport and storage networks. This paper focuses on the role that biosecurity is set to play in future food security by preventing both pre- and post-harvest losses, thereby protecting crop yield. We model biosecurity as a food security technology that may complement conventional yield improvement policies if the gains in global farm profits are sufficient to offset the costs of implementation and maintenance. Using phytosanitary measures that slow global spread of the Ug99 strain of wheat stem rust as an example of pre-border biosecurity risk mitigation and combining it with post-border surveillance and invasive alien species control efforts, we estimate global farm profitability may be improved by over US$4.5 billion per annum.

The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249) for an alanine abolished the enzyme's catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family.