Relative activity of free NemA_Ec <i>versus</i> PHA bead immobilized NemA_Ec with different substrates.

<p>Replicate reactions were established containing 200 µM NADPH; 10 µl of either 0.34 µg.ml⁻¹ purified NemA_Ec or NemA_Ec PHA bead suspension; and 100 µM of either K₂CrO₄, <i>N</i>-ethylmaleimide, or CB1954. After 10 min incubation at 22°C the concentration of each substrate remaining was measured, enabling the concentration of substrate reduced to be calculated. Data are the mean of three independent replicates, and error bars indicate ±1 standard deviation.</p

Cr(VI) reduction by NemA_Ec in the presence or absence of a cofactor regenerating partner.

<p>15 µg NemA_Ec were incubated at 22°C with 150 µM potassium chromate in 50 mM sodium phosphate buffer (pH 7.0) and either excess NADH (1 mM; ▪), limiting NADH (50 µM; •), 50 µM NAD⁺ (▴), or 50 µM NAD⁺ together with 15 µg FDH and 5 mM formic acid (♦). Reactions were initiated by addition of NemA_Ec and the concentration of Cr(VI) remaining at each of the time-points indicated was measured by diphenyl carbazide assay. Data are the mean of three independent replicates, and error bars indicate ±1 standard deviation.</p

Cr(VI) transformation by PHA beads displaying functional NemA_Ec fused to covalently tethered PhaC from <i>R. eutropha</i>.

<p>65 µg of PHA beads displaying NemA_Ec were incubated with 150 µM potassium chromate, 15 µg FDH, 5 mM formic acid and 50 µM NAD⁺ in 0.5 ml of 100 mM sodium phosphate buffer (pH 7) and incubated at 22°C for 75 min. The concentration of Cr(VI) remaining at each of the time-points indicated was measured by diphenyl carbazide assay. Data are the mean of three independent replicates, and error bars indicate ±1 standard deviation.</p

Effect of adding cell lysate on Cr(VI) reductase activity of purified enzymes.

<p>Reactions contained 150 μM Cr(VI), 500 µM NADH, and 0.03 mg/ml enzyme in a total volume of 100 μl made up with either 100%, 67%, 33% or 0% <i>E. coli</i> cell lysate as indicated in the key (the remaining volume being made up by 50 mM Tris-Cl buffer pH 7.0). Reactions were stopped at 5 min (NemA_Ec) or 30 min (NfsA_Kp and AzoR_Ec) by addition of 1,5-diphenylcarbazide color developing solution, and the amount of Cr(VI) reduced was calculated. Data are the mean of two independent replicates, and error bars indicate ±1 standard deviation.</p

Kinetic parameters for purified recombinant oxidoreductases with Cr(VI) as substrate.

a<p>Enzyme nomenclature is described in full in Materials and Methods. With the exception of NemA_Ec, all enzyme kinetics were measured with NADH as cofactor on the basis that this cofactor is more cost-effective for high level use than NADPH. NemA_Ec kinetic parameters were measured with both cofactors to enable any preference to be identified.</p>b<p>Apparent <i>K_M</i> and <i>k_cat</i> as measured at 1 mM NADH (or 1 mM NADPH, for NemA_Ec only). Data are the mean of two independent replicates, and error bars indicate ±1 standard deviation.</p>c<p>No error bars are available, as derivation of meaningful values from a Lineweaver-Burk plot for these low activity enzymes required the replicate data to be averaged prior to analysis.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Bioactivity-Guided Metabolite Profiling of Feijoa (<i>Acca sellowiana</i>) Cultivars Identifies 4‑Cyclopentene-1,3-dione as a Potent Antifungal Inhibitor of Chitin Synthesis

Pathogenic fungi continue to develop resistance against current antifungal drugs. To explore the potential of agricultural waste products as a source of novel antifungal compounds, we obtained an unbiased GC-MS profile of 151 compounds from 16 commercial and experimental cultivars of feijoa peels. Multivariate analysis correlated 93% of the compound profiles with antifungal bioactivities. Of the 18 compounds that significantly correlated with antifungal activity, 5 had not previously been described from feijoa. Two novel cultivars were the most bioactive, and the compound 4-cyclopentene-1,3-dione, detected in these cultivars, was potently antifungal (IC₅₀ = 1–2 μM) against human-pathogenic <i>Candida</i> species. Haploinsufficiency and fluorescence microscopy analyses determined that the synthesis of chitin, a fungal-cell-wall polysaccharide, was the target of 4-cyclopentene-1,3-dione. This fungal-specific mechanism was consistent with a 22–70-fold reduction in antibacterial activity. Overall, we identified the agricultural waste product of specific cultivars of feijoa peels as a source of potential high-value antifungal compounds