Flavin-dependent halogenases involved in secondary metabolism in bacteria

The understanding of biological halogenation has increased during the last few years. While haloperoxidases were the only halogenating enzymes known until 1997, it is now clear that haloperoxidases are hardly, if at all, involved in biosynthesis of more complex halogenated compounds in microorganisms. A novel type of halogenating enzymes, flavin-dependent halogenases, has been identified as a major player in the introduction of chloride and bromide into activated organic molecules. Flavin-dependent halogenases require the activity of a flavin reductase for the production of reduced flavin, required by the actual halogenase. A number of flavin-dependent tryptophan halogenases have been investigated in some detail, and the first three-dimensional structure of a member of this enzyme subfamily, tryptophan 7-halogenase, has been elucidated. This structure suggests a mechanism involving the formation of hypohalous acid, which is used inside the enzyme for regioselective halogenation of the respective substrate. The introduction of halogen atoms into non-activated alkyl groups is catalysed by non-heme Fe^II α-ketoglutarate- and O₂-dependent halogenases. Examples for the use of flavin-dependent halogenases for the formation of novel halogenated compounds in in vitro and in vivo reactions promise a bright future for the application of biological halogenation reactions.     

黄素依赖型卤化酶的结构特点及工程改造*

卤化物是通过卤化酶催化卤族元素在有机化合物上特定位置发生取代形成的一类化合物,具有独特的生理生化作用。黄素依赖型卤化酶具有良好的区域选择性,虽然有相似的黄素分子的结合位点,但在底物结合方面略有不同,对其结构和合成途径及结合蛋白质工程的随机诱变和定向改造的研究在工业应用中至关重要。讨论了具有高区域选择性的黄素依赖型卤化酶的结构特点及工程改造,以及经过工程改造后黄素依赖型卤化酶在工业生产中的应用。     

Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases

Numerous polyketides are known from bacteria, plants, and fungi. However, only a few have been isolated from basidiomycetes. Large scale genome sequencing projects now help anticipate the capacity of basidiomycetes to synthesize polyketides. In this study, we identified and annotated 111 type I and three type III polyketide synthase (PKS) genes from 35 sequenced basidiomycete genomes. Phylogenetic analysis of PKS genes suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged. A comparison of genomic and metabolomic data shows that the number of polyketide genes exceeds the number of known polyketide structures by far. Exploiting these results to design degenerate PCR primers, we amplified and cloned the complete sequence of armB, a PKS gene from the melleolide producer Armillaria mellea. We expect this study will serve as a guide for future genomic mining projects to discover structurally diverse mushroom-derived polyketides.     

Structural Insights into Regioselectivity in the Enzymatic Chlorination of Tryptophan

The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom. Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family. These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position. We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl⁻, O₂, and 1,5-dihydroflavin adenine dinucleotide. We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module. The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role. This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily.     

Response of mycorrhizal grapevine to Armillaria mellea inoculation: disease development and polyamines

A study was conducted with the vine rootstock Richter 110 (Vitis berlandieri Planch. x Vitis rupestris L.) in order to assess whether the colonisation by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices (BEG 72) can delay the disease development in plants inoculated with the root-rot fungus Armillaria mellea (Vahl:Fr) Kummer, and to elucidate if the levels of polyamines (PAs) are modified in response to G. intraradices, A. mellea or by the dual infection. Four treatments were considered: control and G. intraradices-inoculated plants infected or not with A. mellea. Plant growth, mycorrhizal colonisation and disease development were monitored throughout the experiment. High performance liquid chromatography (HPLC) in combination with fluorescence spectrophotometry was used to separate and quantify free root and leaf polyamines. The slower development of pathogenic symptoms and the higher plant biomass of mycorrhizal plants inoculated with A. mellea indicate an increase of tolerance due to the AMF inoculation. The variations in free PA levels detected at the beginning of the pathogenic infection suggest that PAs may have a potential role in the signalling mechanisms of the tolerance of mycorrhizal plants against A. mellea.     

Why does the bioluminescent fungus <Emphasis Type="Italic">Armillaria mellea</Emphasis> have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin—the key component of fungal bioluminescent system—was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Discovery of a novel series of hDHODH inhibitors with anti-pulmonary fibrotic activities

Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme essential to pyrimidine de novo biosynthesis, which serves as an attractive therapeutic target for the treatment of autoimmune disorders. A novel series of hDHODH inhibitors was developed based on a lead which was obtained by a medicinal chemistry exploration. Most compounds showed moderate to significant potency against hDHODH, compounds 5d, 5e, and 6a effectively inhibited the activities of hDHODH with IC₅₀ values from 0.9 to 2.8 μM. Further studies showed that compound 5e also effectively suppressed proliferation of the activated PBMCs (IC₅₀ = 20.35 μM). Surprisingly, compound 5e also showed anti-pulmonary fibrotic activity similar to that of pirfenidone in vitro assay. Therefore, compound 5e might have potential to be developed as a novel hDHODH inhibitors for autoimmune diseases therapy.     

Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.     

Recent advances in the development of biosensor for phenol: a review

Phenol and its derivatives are widespread contaminants whose sources are both natural and industrial. Phenol is massively produced and used as a starting material for synthetic polymers and fibers. Although phenolic compounds play important biochemical and physiological roles in living systems, their accumulation in the environment as a result of intensive human activity may result in drastic ecological problem. Various analytical techniques are available for the detection of phenol in environmental samples. But they need complex sample pre-treatment so as are time consuming, costly and use heavy devices. On the other hand a biosensor is a device that gives rapid detection, cost effective and easy. A review study was carried out to accumulate the possible biosensors for the detection of phenolic compounds in environmental samples. A number of biological components including microorganisms, enzymes, antibodies, antigens, nucleic acids etc. can be used for the construction of biosensors that was found to detect phenolic compounds. Of all type of biological components microorganisms and enzymes are mostly used. The microorganisms are Pseudomonas, Moraxella, Arthrobacter, Rhodococcus, and Trichosporon. The most used enzymes are tyrosinase, peroxidase, laccase, glucose dehydrogenase, cellobiose dehydrogenase etc. Antibody sensors can detect a very trace level. The biorecognition of DNA biosensors occur by hybridization of DNA. Biosensors are found to work well when the biological sensing element is immobilized. A variety of immobilization techniques were found to use as adsorption, covalent binding, entrapment, cross-linking etc. For immobilization the matrices used was polyvinyl alcohol, Osmium complex, nafion/sol?Cgel silicate, chitosan, silica gel etc.     

Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.     

Identification of low micromolar dual inhibitors for aldose reductase (ALR2) and poly (ADP-ribose) polymerase (PARP-1) using structure based design approach

Clinical studies have revealed that diabetic retinopathy is a multifactorial disorder. Moreover, studies also suggest that ALR2 and PARP-1 co-occur in retinal cells, making them appropriate targets for the treatment of diabetic retinopathy. To find the dual inhibitors of ALR2 and PARP-1, the structure based design was carried out in parallel for both the target proteins. A series of novel thiazolidine-2,4-dione (TZD) derivatives were therefore rationally designed, synthesized and their in vitro inhibitory activities against ALR2 and PARP-1 were evaluated. The experimental results showed that compounds 5b and 5f, with 2-chloro and 4-fluoro substitutions, showed biochemical activities in micromolar and submicromolar range (IC₅₀ 1.34–5.03 μM) against both the targeted enzymes. The structure-activity relationship elucidated for these novel inhibitors against both the enzymes provide new insight into the binding mode of the inhibitors to the active sites of enzymes. The positive results of the biochemical assay suggest that these compounds may be further optimized and utilized for the treatment of diabetic retinopathy.     

The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur

Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.     

Rhizomorph Behaviour in Armillaria mellea (Fr.) Quel.: With one Figure in the text: Saprophytic Colonization of Woody Substrates in Soil

Rhizomorphs put out by Armillaria mellea from small woody inoculain glass tubes of moist soil were tested for their ability tocolonize segments of willow shoots, previously killed by autoclavingand then buried for various periods in soil before inoculationwith A. mellea. The degree of saprophytic colonization of thesesubstrate segments by A. mellea was assessed by reburying themin fresh tubes of moist soil, and recording weekly growth incrementsof rhizomorphs put out from them over a period of 5 weeks. Aperiod of previous burial in soil up to 3 weeks was found notto diminish availability of the substrate segments to A. mellea,but with longer periods substrate value for A. mellea progressivelydeclined. Segments of living, green shoots of willow provideda consistently better substrate for A. mellea than did deadsegments, and these living segments maintained their substratevalue over periods (up to 7 weeks) of previous burial in soil.These results are interpreted in terms of competition betweenA. mellea and other soil fungi. In the infection of living tissues,A. mellea benefits from the exclusion of competitors by hostresistance. In the saprophytic colonization of dead tissues,A. mellea has the advantage of its capacity to decompose celluloseand lignin, which are substrates restricted to a minority ofsoil fungi.     

Characterisation of the secreted apyrase family of Heligmosomoides polygyrus

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.     

Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding,Organization of Active Site Residues and Stereopreferences

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.     

富硒条件下蜜环菌菌株硒耐受性及胞外酶生物活性的研究

[目的]通过对蜜环菌(Armillaria mellea)的富硒驯化,研究各菌株硒耐受性、有机硒含量及生物活性的变化规律,从而获得生物活性更强的蜜环菌,并对富硒蜜环菌的生物学特征进行初步研究.[方法]以Na2SeO3为无机硒试剂对蜜环菌进行富硒驯化;采用氢化物原子荧光光谱法测定蜜环菌的硒含量,热水浴法测定蜜环菌的无机硒...     

Anti-Zika candidates from a marine fungus with a remarkable biosynthetic repertoire

The study of natural products provides exciting opportunities for the discovery of novel biologically active molecules and biosynthetic pathways. Recently, Yuan and colleagues described 30 cyclic depsipeptides that are biosynthesized by proteins encoded by three distinct gene clusters in the marine fungus, Beauveria felina. Genetic and biochemical studies confirmed the involvement of nonribosomal peptide synthetases in the production of multiple compounds, some of which inhibit Zika virus replication.

Recent outbreaks caused by Zika virus, a member of the positive strand RNA flavivirus genus, have caused alarm notably due to the transmissibility of the virus across the placental barrier. Vaccines against Zika virus are still under development, and there are currently no approved antiviral treatments. Microbial natural products are an important source and inspiration for the development of novel pharmaceuticals, including as antiviral compounds (1). Many natural products originally identified during a period of in-depth investigation between the 1960s and 1980s are now being reexamined following advances in microbial culturing and genomic sequencing, as well as advances in biochemical and heterologous methods for production of these compounds. During the past decade, our understanding of the biosynthesis pathways that produce these compounds has increased, helping to realize the long-standing goal of predicting products from gene sequences and engineering or combining catalysts from different biosynthetic gene clusters to produce novel compounds with desirable activities (2).Many peptide natural products are produced by the nonribosomal peptide synthetases (NRPSs), a family of megasynthetase proteins that produce peptides using an unusual enzymatic architecture. Generally, NRPSs consist of fused catalytic domains that function with an assembly line strategy to produce a variety of peptide natural products, including antibiotics, siderophores, signaling molecules, toxins, and antitumor molecules (3). Independent of ribosomes, mRNAs, and tRNAs, the NRPS enzymes can incorporate nonproteinogenic amino acids into their products. An important class of NRPS products is the cyclodepsipeptides (CDPs), which are cyclic arrangements of amino and hydroxy acids joined with amide and ester bonds (4). One family of cyclohexadepsipeptides includes destruxins (DTXs), isaridins (ISDs), and isariins (ISRs), which all contain six building blocks, including one β-amino or β-hydroxy acid, to form a 19-membered macrocycle. Multiple DTX, ISD, and ISR analogs exhibit diverse insecticidal, antifungal, antiviral, and antibacterial activities, and understanding the enzymatic basis of CDP biosynthesis can provide opportunities to develop new drugs with enhanced activities.In a recent article in the Journal of Biological Chemistry, Yuan et al. (5) reported the discovery and characterization of biosynthetic gene clusters of several CDPs from the sea-sponge-associated fungus Beauveria felina. As part of an ongoing antiviral discovery campaign, a crude extract Beauveria was found to exhibit anti-Zika virus activity. The extract was subjected to NMR analysis as well as classification via the Global Natural Product Social (GNPS) molecular network resource (6), which serves as a public database for reference mass spectrometry data with crowd-sourced annotation. The clustering of observed spectra identified CDPs similar—and in some cases identical—to previously identified DTXs, ISDs, and ISRs. A total of 30 unique CDPs were identified and isolated, harboring nonproteinogenic amino acid building blocks, including β-alanine, (3S)-methyl-L-proline (3MP), and a variety of α-hydroxy acids such as α-D-hydroxyisocaproic acid (HIC) that form the single ester linkage of each CDP. Twenty-six of the compounds were shown to have low cytotoxicity against a lung epithelial cell line and were therefore tested in a Zika virus replication model. Seven active compounds had a significant inhibitory effect on viral replication as monitored by quantification of viral RNA and the production of an abundant viral nonstructural protein. A subset of these compounds was further examined and shown to be effective at an early stage of viral replication, potentially by blocking endosome acidification. Curiously, six of the seven active compounds contained modified HIC residues, suggesting this may be important for the observed antiviral activity.The B. felina genome sequence reported by Yuan and colleagues identified 23 genes that encode NRPS proteins, three of which contain the six modules necessary for the production of CDPs containing six building blocks (Fig. 1). Bioinformatic analysis and gene deletion studies allowed the authors to assign all of the CDP products to the responsible biosynthetic gene clusters. Biosynthetic pathways were described to produce the different analogs from each cluster, and experimental evidence was provided to confirm the necessary auxiliary enzymes for producing 3MP and for modifying the HIC residues. 3MP has been observed in other nonribosomal and ribosomally encoded peptides, which respectively form the 3MP residue before or after peptide formation. In the production of the Beauveria CDPs, the Fe/α-ketoglutarate-dependent oxygenase DetxE was confirmed via gene deletion and biochemical assays to convert isoleucine to 4-methyl-Δ¹-pyrroline-5-carboxylate. The final conversion to 3MP was again demonstrated with both genetic and biochemical experiments, which implicated one of two Beauveria Δ¹-pyrroline-5-carboxylate reductases (P5CRs) in the formation of 3MP. B. felina therefore exploits the enzymes from primary metabolism to expand the diversity of CDPs that are produced. Understanding the rules that enable some organisms to incorporate enzymes from other pathways will expand our ability to predict products from genome sequences and engineer the production of novel compounds through heterologous or systems biology approaches.Open in a separate windowFigure 1The marine sponge-associated fungus B. felina uses three biosynthetic pathways that involve large modular NRPS enzymes to produce as many as 30 cyclohexadepsipeptides, harboring nonproteinogenic amino acids and α-hydroxyacids. Several compounds showed promising anti-Zika virus activity.The modular nature of the NRPS enzymes has raised the possibility that novel enzyme assembly lines could be designed by mutating or rearranging specific domains or modules. Recent structures of multidomain NRPS enzymes have fostered exciting progress in these efforts (7). In particular, the identification of tractable junction points for combinatorial engineering has enabled the formation of hybrid NRPSs from closely related systems to produce novel peptide products at high titres in several model systems (8, 9). The similarity of the fungal CDP systems provides an opportunity to diversify further the depsipeptide structures and produce additional compounds. Moreover, the reported biosynthesis of 3MP illustrates how a deeper understanding of the interactions of primary and secondary metabolic pathways in natural product biosynthesis can perhaps be used to incorporate necessary catalytic steps to expand the diversity of final products or improve the yield of important compounds. The mechanistic importance of the methyl group of the 3MP is as yet not known.It is noteworthy that the majority of the compounds that prove most promising in the anti-Zika assays were destruxins that contain tailoring modifications that appear to be initiated by an oxidation by a Cytochrome P450 at the HIC residue (5). The mechanistic basis of this antiviral activity remains unknown; however, potential modifications to other inactive CDPs using either the B. felina P450 or other heterologous catalysts may identify additional compounds with desired activities. This recent study from Yuan et al. (10) expands the arsenal of biologically active natural products from the sponge-associated microbiome, which includes both ribosomally derived and nonribosomal peptides. These studies should inspire future screening of natural products, microbial culturing, and genome mining of unique microbes such as sponge-associated fungi or uncultured bacteria from diverse environments to discover novel natural products or new derivatives of known compounds with greater efficacy.     

Chlorella vulgaris Aldehyde Reductase Is Capable of Functioning as Ferric Reductase and of Driving the Fenton Reaction in the Presence of Free Flavin

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.