Identification of a family of species-selective complex I inhibitors as potential anthelmintics.

Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.

A facile cellulose finishing strategy through in-situ growth of sliver-doped manganese dioxide assisted by amine-quinone for improving indoor living quality.

Nowadays, various harmful indoor pollutants especially including bacteria and residual formaldehyde (HCHO) seriously threaten human health and reduce the quality of public life. Herein, a universal substrate-independence finishing approach for efficiently solving these hybrid indoor threats is demonstrated, in which amine-quinone network (AQN) was employed as reduction agent to guide in-situ growth of Ag@MnO2 particles, and also acted as an adhesion interlayer to firmly anchor nanoparticles onto diverse textiles, especially for cotton fabrics. In contrast with traditional hydrothermal or calcine methods, the highly reactive AQN ensures the efficient generation of functional nanoparticles under mild conditions without any additional catalysts. During the AQN-guided reduction, the doping of Ag atoms onto cellulose fiber surface optimized the crystallinity and oxygen vacancy of MnO2, providing cotton efficient antibacterial efficiency over 90 % after 30 min of contact, companying with encouraging UV-shielding and indoor HCHO purification properties. Besides, even after 30 cycles of standard washing, the Ag@MnO2-decorated textiles can effectively degrade HCHO while well-maintaining their inherent properties. In summary, the presented AQN-mediated strategy of efficiently guiding the deposition of functional particles on fibers has broad application prospects in the green and sustainable functionalization of textiles.

In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Quinones-enhanced humification in food waste composting: A novel strategy for hazard mitigation and nitrogen retention.

The harmless and high-value conversion of organic waste are the core problems to be solved by composting technology. This study introduced an innovative method of promoting targeted humification and nitrogen retention in composting by adding p-benzoquinone (PBQ), the composting without any additives was set as control group (CK). The results indicated that the addition of exogenous quinones led to a 30.1% increase in humic acid (HA) content during the heating and thermophilic phases of composting. Spectroscopic analyses confirmed that exogenous quinones form the core skeleton structure of amino-quinones in HA through composting biochemical reactions. This accelerated the transformation of quinones into recalcitrant HA in the early stages of composting, and reduced CO2 and NH3 by 8% and 78%, respectively. Redundancy analysis (RDA) revealed that the decrease in carbon and nitrogen losses primarily correlated with quinones enhancing HA formation and greater nitrogen incorporation into HA (P < 0.05). Furthermore, the compost treated with quinones demonstrated a decrease in phytotoxicity and earthworm mortality, alongside a significant increase in the relative abundance of actinobacteria, which are associated with the humification process. This research establishes and proposes that co-composting with quinones-containing waste is an effective approach for the sustainable recycling of hazardous solid waste.

Electrochemical Analysis for Total Alkalinity of Water by the Measurement of Cathodic Prepeak of Quinone Caused by Surplus Acid.

In this study, an electrochemical analysis, coupled with the concept of back neutralization titration and the voltammetric determination of surplus acid, is proposed for determining the total alkalinity of water samples. When linear sweep voltammetry of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ) with H2SO4 in a water and ethanol (44 : 56, v/v) mixture was carried out using a bare glassy carbon working electrode, a cathodic prepeak of DBBQ caused by H2SO4 was observed on the voltammogram at a more positive potential than when compared with the original cathodic peak of DBBQ. When similar voltammetry was carried out in the presence of Na2CO3 and H2SO4, the cathodic prepeak height of DBBQ was decreased with an increase in the Na2CO3 concentration. The decrease of the cathodic prepeak height of DBBQ was found to be linearly related to the Na2CO3 concentration ranging from 0.025 to 2.5 mM (r2 = 0.998). The total equivalent concentrations of inorganic bases in samples of mineral water and tap water were determined, and then the results were converted to the total alkalinities of the water samples (mg/L CaCO3). The total alkalinities of the water samples determined by the present electrochemical analysis were essentially the same compared with those by the neutralization titration method. From these results, we were able to demonstrate that the present electrochemical analysis with accuracy and precision could be applied to determine the total alkalinity, which is one of the indicators to examine water quality. The present electrochemical analysis would contribute to achieving the sustainable development goals (SDGs) of #6 and #14.

A novel L-shaped ortho-quinone analog as PLK1 inhibitor blocks prostate cancer cells in G2 phase.

Prostate cancer is the most common malignant tumor among men worldwide. Currently, the main treatments are radical prostatectomy, radiotherapy, chemotherapy, and endocrine therapy. However, most of them are poorly effective and induce side effects. Polo-like kinase 1 (PLK1) regulates cell cycle and mitosis. Its inhibitor BI2536 promotes the therapeutic effect of nilotinib in chronic myeloid leukemia, enhances the sensitivity of neural tube cell tumors to radiation therapy and PLK1 silencing enhances the sensitivity of squamous cell carcinoma to cisplatin. Therefore, the aim of this study was to evaluate the effect of the PLK1 inhibitor L-shaped ortho-quinone analog TE6 on prostate cancer. In vitro on prostate cancer cells showed that TE6 inhibited PLK1 protein expression and consequently cell proliferation by blocking the cell cycle at G2 phase. In vivo on a subcutaneous tumor model in nude mice confirmed that TE6 effectively inhibited tumor growth in nude mice, inhibited PLK1 expression and regulated the expression of cell cycle proteins such as p21, p53, CDK1, Cdc25C, and cyclinB1. Thus, PLK1 was identified as the target protein of TE6, these results reveal the critical role of PLK1 in the growth and survival of prostate cancer and point out the ability of TE6 on targeting PLK1, being a potential drug for prostate cancer therapy.

Pseudoceranoids A-J, Sesquiterpene-Based Meroterpenoids with Cytotoxicity from the Sponge Pseudoceratina purpurea.

Pseudoceranoid A (1), a rare merosesquiterpene featuring a rearranged 4,9-friedodrimane-type core with a crotonolactone moiety, two new rearranged 4,9-friedodrimane-type sesquiterpene cyclopentanones (2 and 3), and three new rearranged 4,9-friedodrimane-type sesquiterpene hydroquinones (4-6), along with two new drimane-type sesquiterpene derivatives (7 and 8), as well as two new 4,9-friedodrimane-type sesquiterpene quinones (9 and 10), were isolated from the South China Sea sponge Pseudoceratina purpurea. The structures of compounds were established by analysis of spectroscopic data, as well as by single-crystal X-ray diffraction, DP4+ probability analyses, and calculated electronic circular dichroism. Compound 4 showed weak cytotoxicity against K562, H69AR, and MDAMB-231 cell lines with IC50 values of 3.01, 7.74, and 9.82 µM, respectively. Compound 5 exhibited cytotoxicity against the H69AR cell line with an IC50 value of 2.85 µM.

Quinones: Biosynthesis, Characterization of 13 C Spectroscopical Data and Pharmacological Activities.

Quinones are natural products widely distributed in nature, which are involved in stages of several vital biological processes, with mostly having a variety of pharmacological properties. The main groups comprising most of these compounds are benzoquinones, naphthoquinones, anthraquinones, and phenanthraquinones. Quinone isolation has been a focus of study around the world in recent years; for this reason, this study approaches the junction of natural quinones identified by 13 C Nuclear Magnetic Resonance (NMR) spectroscopic analytical techniques. The methodology used to obtain the data collected articles from various databases on quinones from 2000 to 2022. As a result, 137 compounds were selected, among which 70 were characterized for the first time in the period investigated; moreover, the study also discusses the biosynthetic pathways of quinones and the pharmacological activities of the compounds found, giving an overview of the various applications of these compounds.

A trick of the tail: computing the entropic contribution to the energetics of quinone-protein unbindung.

We estimate the entropic contributions to the free energy of quinone unbinding in bacterial and mitochondrial respiratory chains using molecular dynamics (MD) and Monte Carlo (MC) computer simulations. For a varying length of the isoprenoid side chain, MD simulations in lipid bilayers and in unpolar solvents are used to assess the dihedral angle distributions along the chain. These form the basis of a MC estimate of the number of molecular structures that do not exhibit steric self-overlap and that are confined to the bilayer. We obtain an entropy drive of TΔS = 1.4 kcal mol-1 for each isoprene unit, which in sum is comparable to the redox potential differences involved in respiratory chain electron transfer. We postulate an entropy-driven zipper for quinone unbinding and discuss it in the context of the bioenergetics and the structure of complex I, and we indicate possible consequences of our findings for MD-based free energy computations.

Quantification of Quinones in Environmental Media by Chemical Tagging with Cysteine-Containing Peptides Coupled to Size Exclusionary Separation.

Quinones are one of the most important redox-reactive organic compounds in natural environments, such as soil, water, and sediment, playing an important role in regulating the environmental processes and biogeochemical cycles of critical elements under climate change, including the influences of extreme events such as wildfires. However, to date, no existing methods can quantify quinones in complex environmental media. To overcome this challenge, a quantification method was developed by coupling chemical tagging of quinones by cysteine-containing nonaromatic peptides (Cpep) through a Michael addition reaction with size exclusionary chromatography (SEC) separation and ultraviolet (UV) analysis─leveraging on the characteristic absorbance of aromatic rings at 254 nm and molecular size of peptide. The method was demonstrated using model quinones, including 1,4-benzoquinone (BQ), 1,4-naphthoquinone (NQ), and 1,4-anthraquinone (AQ), with a detection limit of 3.3, 0.7, and 0.2 µM, respectively. Concentrations of quinones in water extractions of biochars, soils, and wildfire-derived ashes were determined to range from 0.8 to 14 µM and were positively correlated with their redox reactivity determined by a chemical assay. This method provides a novel rapid quantification of quinones in complex environmental media as well as a quick assessment for redox reactivity and opens up new avenues for studying environmental transformation and remediation of contaminants.

Probing molecular interactions of semiquinone radicals at quinone reduction sites of cytochrome bc1 by X-band HYSCORE EPR spectroscopy and quantum mechanical calculations.

Quinone redox reactions involve a semiquinone (SQ) intermediate state. The catalytic sites in enzymes stabilize the SQ state via various molecular interactions, such as hydrogen bonding to oxygens of the two carbonyls of the benzoquinone ring. To understand how these interactions contribute to SQ stabilization, we examined SQ in the quinone reduction site (Qi) of cytochrome bc1 using electron paramagnetic resonance (ESEEM, HYSCORE) at the X-band and quantum mechanical (QM) calculations. We compared native enzyme (WT) with a H217R mutant (replacement of histidine that interacts with one carbonyl of the occupant of Qi to arginine) in which the SQ stability has previously been shown to markedly increase. The 14N region of the HYSCORE 2D spectrum for SQi in WT had a shape typical of histidine residue, while in H217R, the spectrum shape changed significantly and appeared similar to the pattern described for SQ liganded natively by arginine in cytochrome bo3. Parametrization of hyperfine and quadrupolar interactions of SQi with surrounding magnetic nuclei (1H, 14N) allowed us to assign specific nitrogens of H217 or R217 as ligands of SQi in WT and H217R, respectively. This was further substantiated by qualitative agreement between the experimental (EPR-derived) and theoretical (QM-derived) parameters. The proton (1H) region of the HYSCORE spectrum in both WT and H217R was very similar and indicative of interactions with two protons, which in view of the QM calculations, were identified as directly involved in the formation of a H-bond with the two carbonyl oxygens of SQ (interaction of H217 or R217 with O4 and D252 with O1). In view of these assignments, we explain how different SQ ligands effectively influence SQ stability. We also propose that the characteristic X-band HYSCORE pattern and parameters of H217R are highly specific to the interaction of SQ with the nitrogen of arginine. These features can thus be considered as potential markers of the interaction of arginine with SQ in other proteins.

Heterocyclic Iminoquinones and Quinones from the National Cancer Institute (NCI, USA) COMPARE Analysis.

This review uses the National Cancer Institute (NCI) COMPARE program to establish an extensive list of heterocyclic iminoquinones and quinones with similarities in differential growth inhibition patterns across the 60-cell line panel of the NCI Developmental Therapeutics Program (DTP). Many natural products and synthetic analogues are revealed as potential NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates, through correlations to dipyridoimidazo[5,4-f]benzimidazoleiminoquinone (DPIQ), and as potential thioredoxin reductase (TrxR) inhibitors, through correlations to benzo[1,2,4]triazin-7-ones and pleurotin. The strong correlation to NQO1 infers the enzyme has a major influence on the amount of the active compound with benzo[e]perimidines, phenoxazinones, benz[f]pyrido[1,2-a]indole-6,11-quinones, seriniquinones, kalasinamide, indolequinones, and furano[2,3-b]naphthoquinones, hypothesised as prodrugs. Compounds with very strong correlations to known TrxR inhibitors had inverse correlations to the expression of both reductase enzymes, NQO1 and TrxR, including naphtho[2,3-b][1,4]oxazepane-6,11-diones, benzo[a]carbazole-1,4-diones, pyranonaphthoquinones (including kalafungin, nanaomycin A, and analogues of griseusin A), and discorhabdin C. Quinoline-5,8-dione scaffolds based on streptonigrin and lavendamycin can correlate to either reductase. Inhibitors of TrxR are not necessarily (imino)quinones, e.g., parthenolides, while oxidising moieties are essential for correlations to NQO1, as with the mitosenes. Herein, an overview of synthetic methods and biological activity of each family of heterocyclic imino(quinone) is provided.

Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site 2: Identification of neutral state quinone bands.

Time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study photosystem I (PSI) from Synechocystis sp. PCC 6803 with four high-potential, 1,4-naphthoquinones (NQs) incorporated into the A1 binding site. The incorporated quinones are 2-chloro-NQ (2ClNQ), 2-bromo-NQ (2BrNQ), 2,3-dichloro-NQ (Cl2NQ), and 2,3-dibromo-NQ (Br2NQ). For completeness 2-methyl-NQ (2MNQ) was also incorporated and studied. Previously, PSI with the same quinones incorporated were studied in the, so-called, anion spectral region between 1550 and 1400 cm-1 (Agarwala et al. in Biochim Biophys Acta 1864(1):148918, 2023). Here we focus on spectra in the previously unexplored 1400-1200 cm-1 spectral region. In this region several bands are identified and assigned to the neutral state of the incorporated quinones. This is important as identification of neutral state quinone bands in the regular 1700-1600 cm-1 region has proven difficult in the past. For neutral PhQ in PSI a broad, intense band appears at ~ 1300 cm-1. For the symmetric di-substituted NQs (Cl2NQ/Br2NQ) a single intense neutral state band is found at ~ 1280/1269 cm-1, respectively. For both mono-substituted NQs, 2ClNQ and 2BrNQ, however, two neutral state bands are observed at ~ 1280 and ~ 1250 cm-1, respectively. These observations from time-resolved spectra agree well with conclusions drawn from absorption spectra of the NQs in THF, which are also presented here. Density functional theory based vibrational frequency calculations were undertaken allowing an identification of the normal modes associated with the neutral state quinone bands.

Covalent adduction of serotonin-derived quinones to the SARS-CoV-2 main protease expressed in a cultured cell.

The SARS-CoV-2 main protease is an essential molecule for viral replication and is often targeted by medications to treat the infection. In this study, we investigated the possible inhibitory action of endogenous quinones on the enzyme. Recombinant SARS-CoV-2 main protease was exposed to tryptamine-4,5-dione (TD) or quinone from 5-hydroxyindoleacetic acid (Q5HIAA). As a result, the protease activity was considerably decreased in a dose-dependent manner. The IC50 values of the quinones toward the enzyme were approximately 0.28 µM (TD) and 0.49 µM (Q5HIAA). Blot analyses using specific antibodies to quinone-modified proteins revealed that quinones were adducted to the enzyme at concentrations as low as 0.12 µM. Intact mass analyses showed that one or two quinone molecules were covalently adducted onto the main protease. Chymotrypsin-digested main protease analyses revealed that the quinones bind to thiol residues at the enzyme's active site. When TD or Q5HIAA were exposed to cultured cells expressing the viral enzyme, quinone-modified enzyme was identified in the cell lysate, suggesting that even extracellularly generated quinones could react with the viral enzyme expressed in an infected cell. Thus, these endogenous quinones could act as inhibitors of the viral enzyme.

Simple Strategy for Benzo[g]chromene Synthesis via a Photochemical Intramolecular Cyclization Reaction.

Photochemical reactions are often a desirable strategy for organic synthesis because they do not require toxic and expensive reagents and produce less waste than thermal reactions. Herein, a facile photochemical strategy is described to synthesize benzo[g]chromene derivatives. This strategy utilizes the photoredox reaction of quinones, which allows C-H bond oxidation in the vicinity of the photoexcited quinone carbonyl group. The reaction mechanism was investigated using 1H NMR analysis. The intramolecular cyclization reaction proceeded via the formation of 1,3-dioxole compounds as intermediates by the photoredox reaction of p-quinone, followed by re-cyclization. The synthesized benzo[g]chromene derivatives are important heterocyclic skeletons with useful biological and pharmacological properties.

From polyphenol to o-quinone: Occurrence, significance, and intervention strategies in foods and health implications.

Polyphenol oxidation is a chemical process impairing food freshness and other desirable qualities, which has become a serious problem in fruit and vegetable processing industry. It is crucial to understand the mechanisms involved in these detrimental alterations. o-Quinones are primarily generated by polyphenols with di/tri-phenolic groups through enzymatic oxidation and/or auto-oxidation. They are highly reactive species, which not only readily suffer the attack by nucleophiles but also powerfully oxidize other molecules presenting lower redox potentials via electron transfer reactions. These reactions and subsequent complicated reactions are capable of initiating quality losses in foods, such as browning, aroma loss, and nutritional decline. To attenuate these adverse influences, a variety of technologies have emerged to restrain polyphenol oxidation via governing different factors, especially polyphenol oxidases and oxygen. Despite tremendous efforts devoted, to date, the loss of food quality caused by quinones has remained a great challenge in the food processing industry. Furthermore, o-quinones are responsible for the chemopreventive effects and/or toxicity of the parent catechols on human health, the mechanisms by which are quite complex. Herein, this review focuses on the generation and reactivity of o-quinones, attempting to clarify mechanisms involved in the quality deterioration of foods and health implications for humans. Potential innovative inhibitors and technologies are also presented to intervene in o-quinone formation and subsequent reactions. In future, the feasibility of these inhibitory strategies should be evaluated, and further exploration on biological targets of o-quinones is of great necessity.

Structural insights into the action mechanisms of artificial electron acceptors in photosystem II.

Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with molecular structures similar to that of plastoquinone can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95 to 2.10 Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone-binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII and also provide information for the design of more efficient electron acceptors.

It Takes Two to Tango, Part II: Synthesis of A-Ring Functionalised Quinones Containing Two Redox-Active Centres with Antitumour Activities.

In 2021, our research group published the prominent anticancer activity achieved through the successful combination of two redox centres (ortho-quinone/para-quinone or quinone/selenium-containing triazole) through a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The combination of two naphthoquinoidal substrates towards a synergetic product was indicated, but not fully explored. Herein, we report the synthesis of 15 new quinone-based derivatives prepared from click chemistry reactions and their subsequent evaluation against nine cancer cell lines and the murine fibroblast line L929. Our strategy was based on the modification of the A-ring of para-naphthoquinones and subsequent conjugation with different ortho-quinoidal moieties. As anticipated, our study identified several compounds with IC50 values below 0.5 µM in tumour cell lines. Some of the compounds described here also exhibited an excellent selectivity index and low cytotoxicity on L929, the control cell line. The antitumour evaluation of the compounds separately and in their conjugated form proved that the activity is strongly enhanced in the derivatives containing two redox centres. Thus, our study confirms the efficiency of using A-ring functionalized para-quinones coupled with ortho-quinones to obtain a diverse range of two redox centre compounds with potential applications against cancer cell lines. Here as well, it literally takes two for an efficient tango!

Investigation of the Quinone-quinone and Quinone-catechol products using 13C labeling, UPLC-Q-TOF/MS and UPLC-Q-Exactive Orbitrap/MS.

Quinones are highly reactive oxidants and play an essential role in inducing quality deterioration of fruit and vegetable products. Here, a novel stable isotope-labeling approach in combination with high-resolution tandem mass spectrometry UPLC-Q-TOF/MS and UPLC-Q-Exactive Orbitrap/MS, was successfully applied in tracking quinone reaction pathways in both real wines and model reaction systems. Unexpectedly, the binding products of quinone-quinone and quinone-catechol that are not derived from either nucleophilic reaction or redox reaction were discovered and showed the significant high peak area.Self-coupling reactions of semiquinone radicals might provide a possible interpretation for the formation of quinone-quinone products, and a charge transfer reaction coupled with a complementary donor-acceptor interaction is feasibly responsible for the products with a quinone-catechol structure. These findings endow a new perspective for quinone metabolic pathway in foods.

Actinorhodin Biosynthesis Terminates with an Unprecedented Biaryl Coupling Reaction.

A plethora of dimeric natural products exist with diverse chemical structures and biological activities. A major strategy for dimerization is aryl coupling catalyzed by cytochrome P450 or laccase. Actinorhodin (ACT) from Streptomyces coelicolor A3(2) has a dimeric pyranonaphthoquinone structure connected by a C-C bond. In this study, we identified an NmrA-family dimerizing enzyme, ActVA-ORF4, and a cofactor-independent oxidase, ActVA-ORF3, both involved in the last step of ACT biosynthesis. ActVA-ORF4 is a unique NAD(P)H-dependent enzyme that catalyzes the intermolecular C-C bond formation using 8-hydroxydihydrokalafungin (DHK-OH) as the sole substrate. On the other hand, ActVA-ORF3 was found to be a quinone-forming enzyme that produces the coupling substrate, DHK-OH and the final product, ACT. Consequently, the functional assignment of all essential enzymes in the biosynthesis of ACT, one of the best-known model natural products, has been completed.