Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

GC-MS Composition and Olfactory Profile of Concretes from the Flowers of Four Nicotiana Species

The genus Nicotiana (Solanaceae) includes over 70 species, with a long history of traditional use; many of them are nowadays used in bioengineering, biosynthesis, molecular biology, and other studies, while common tobacco, N. tabacum L., is one of the most economically important industrial crops worldwide. Although Nicotiana species have been extensively investigated, relatively less research has focused on flowers, especially research related to obtaining aromatic products for cosmetic and perfumery use. On the other hand, there is evidence that Nicotiana flowers accumulate various secondary metabolites with a distinct aroma and biological activities, and the flowers represent a biomass available in sufficient quantities. Therefore, this study aimed to determinate the chemical composition (by GC-MS) and the olfactory profiles of a specific type of natural aromatic product (concrete), obtained from the flowers of four Nicotiana species, in a direct comparison between them. The yields of extracted concrete were sufficiently high, varying between the species, 1.4% (N. rustica L.), 2.5% (N. glutinosa L.), 1.6% (N. alata Link&Otto genotype with white flowers), 2.7% (N. alata genotype with pink flowers), 3.2% (N. tabacum, Oriental type), and 5.2% (N. tabacum, Virginia type). The major components of the obtained concretes belonged to different chemical classes: N. rustica and N. tabacum (OR), the hydrocarbons n-tetratriacontane (14.5%; 15.0%) and n-triacontane (12.1%; 13.3%), and 3-methyl-pentanoic acid (11.1%; 12.2%); N. glutinosa, the diterpenes sclareol (25.9%), 3-α-hydroxy-manool (16.3%), and 13-epimanool (14.9%); N. alata (WF), the phenylpropanoid terephthalic acid and di(2-ethylhexyl) ester (42.9%); N. alata (PF), the diterpene tributyl acetylcitrate (30.7%); and N. tabacum (FCV), the hydrocarbons n-hexacosane (12.9%) and n-pentacosane (12.9%). Each of the flower concretes revealed a characteristic odor profile. This is the first report about Nicotiana species as a source for obtaining flower concretes; these initial results about the concrete yield, olfactory profile, and chemical composition are a prerequisite for the possible processing of Nicotiana flowers into new aromatic products for use in perfumery and cosmetics. The study provides new data in favor of the potential of the four Nicotiana species as aromatic plants, as well as a possible alternative use of flowers, a valuable, but discarded, plant material in other applications.

An in vitro platform for engineering and harnessing modular polyketide synthases

To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.

Optimizing the Aromatic Product Distribution from Catalytic Fast Pyrolysis of Biomass Using Hydrothermally Synthesized Ga-MFI Zeolites

A series of gallium-containing MFI (Ga-MFI) zeolites with varying Ga2O3/Al2O3 ratios were synthesized using hydrothermal synthesis and tested as catalyst in catalytic fast pyrolysis (CFP) of beech wood for aromatic production. The results show that the incorporation of Ga slightly reduced the effective pore size of Ga-MFI zeolites compared to conventional HZSM-5 zeolites. Therefore, the Ga-MFI zeolites increased the aromatic selectivity for smaller aromatics such as benzene, toluene, and p-xylene and decreased the aromatic selectivity for bulkier ones such as m-xylene, o-xylene, and polyaromatics in CFP of beech wood relative to HSZM-5. In particular, the yield and selectivity of p-xylene, the most desired product from CFP of biomass, increased considerably from 1.64 C% and 33.3% for conventional HZSM-5 to 2.98–3.34 C% and 72.1–79.6% for the synthesized Ga-MFI zeolites. These results suggest that slightly reducing the pore size of MFI zeolite by Ga incorporation has a beneficial effect on optimizing the aromatic selectivity toward more valuable monoaromatic products, especially p-xylene, during CFP of biomass.

Gold-Catalysed Oxidative Cycloisomerisation of 1,6-Diyne Acetates to 1-Naphthyl Ketones

A synthetic method to prepare 1-naphthyl ketones from gold(i)-catalysed oxidative cycloisomerisation of 1,6-diyne acetates is described. The proposed mechanism involves cyclopropenation–cycloreversion of the 1,6-diyne motif initiated by a 1,2-acyloxy migration. This is followed by nucleophilic attack of the ensuing gold carbenoid species by a molecule of water and autoxidation to give the aromatic product.

Phenolic acetals from lignins of varying compositions via iron(iii) triflate catalysed depolymerisation

A small lignin library was used to study the relationship between the lignin structure and the aromatic product yields during acidolysis.

Unusual reactions involved in anaerobic metabolism of phenolic compounds

Aerobic bacteria use molecular oxygen as a common co-substrate for key enzymes of aromatic metabolism. In contrast, in anaerobes all oxygen-dependent reactions are replaced by a set of alternative enzymatic processes. The anaerobic degradation of phenol to a non-aromatic product involves enzymatic processes that are uniquely found in the aromatic metabolism of anaerobic bacteria: (i) ATP-dependent phenol carboxylation to 4-hydroxybenzoate via a phenylphosphate intermediate (biological Kolbe-Schmitt carboxylation); (ii) reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA; and (iii) ATP-dependent reductive dearomatization of the key intermediate benzoyl-CoA in a ‘Birch-like’ reduction mechanism. This review summarizes the results of recent mechanistic studies of the enzymes involved in these three key reactions.

Synthesis and Characterization of a New [4.0.4.0] Porphyrin-like Antiaromatic Macrocycle

The antiaromatic macrocycle 2 has been prepared by the reaction of the bipyrrole dialdehyde 1 with hydrazine. In the crystal structure of 2· DMSO molecule 2 is nearly planar, the mean deviation of the 28 atoms of the core from the best least-squares plane being 0.227 Å. AM1 geometric optimization of the core of 2 leads to a fully planar macrocycle. Attempts to oxidize 2 to 3 were largely unsuccessful, yielding only limited evidence for the formation of 3. The antiaromatic precursor 2 turns out to be much more stable than the putative aromatic product 3. Molecular orbital calculations at the AM1/PM3 level indicate the S 0- T 1 gap for 3 is only about 0.2 eV and hence is readily accessible via intersystem crossing from any visible transition. This small band gap of 3 contrasts starkly with the much larger S 0- T 1 band gap of 1.86 eV calculated for 2 itself. Paratropicity in antiaromatic systems is usually attributed to a low-lying paramagnetic state, with band gaps ranging up to 2.1 eV. For the 2/3 system the S 0- T 1 band gap is substantially larger for the paratropic species than for the putative diatropic species.

The acid-catalyzed demetalation of 1-(tri-n-butylstannyl)-2-phenylethyne. A surprisingly small β-stannyl effect

Rates of conversion of 1-(tri-n-butylstannyl)-2-phenylethyne to phenylethyne in H2O and D2O solutions of perchloric acid were found to be proportional to acid concentration, giving the hydronium ion rate constant [Formula: see text] and the isotope effect [Formula: see text]. The magnitude of this isotope effect suggests that the reaction occurs by rate-determining hydron transfer to the substrate, producing a vinyl carbocation; this carbocation then loses its tributylstannyl group, giving phenylacetylene as the only detectable aromatic product. The hydronium ion rate constant, when compared to the rates of protonation of phenylethyne and 1-(trimethylsilyl)-2-phenylethyne, gives a β-stannyl stabilizing effect of δΔG≠ = 6.6 kcal mol−1 and a differential β-stannyl/β-silyl effect of δΔG≠ = 3.2 kcal mol−1. These stabilizations are very much smaller than previously reported β-stannyl effects. Possible reasons for the difference are suggested. Key words: β-stannyl effect, β-silyl effect, carbocation stabilization, protodemetalation.