INTRODUCTION: Serum albumin (Alb) levels have been found to be independent predictors of all-cause mortality in a community-based population, but whether this is the case for serum cholinesterase (ChE) levels is uncertain. This study aimed to determine whether serum ChE levels are independent predictors of all-cause mortality in a community-based population. METHODS: A total of 3,504 subjects (mean age 62.5 years) from Takahata, Japan participated and were followed up for 13.5 years (median 13.2 years). Based on baseline serum Alb and ChE levels, subjects were stratified by interquartile range as low, middle, and high. The correlation between serum Alb and ChE levels was examined by calculating correlation coefficients. The association between each group and all-cause mortality was examined by Kaplan-Meier and Cox proportional hazards analysis. RESULTS: During follow-up, 568 subjects died. There was a positive correlation between serum Alb and ChE levels (r=0.30). Kaplan-Meier analysis showed that all-cause mortality in the low group was significantly higher for both serum Alb and ChE levels (log-rank P<0.01). Adjusted Cox proportional hazards analysis showed that the serum Alb level was not an independent predictor of all-cause mortality (hazard ratio (HR) 1.18, 95% confidence interval (CI) 0.95-1.46 for all-cause mortality in the low group compared to the middle group), whereas the serum ChE level was an independent predictor of all-cause mortality (HR 1.30, 95% CI 1.06-1.59 for all-cause mortality in the low group compared to the middle group). CONCLUSION: The serum ChE level is an independent predictor of all-cause mortality in the general community-based population.
Gregatin A (1) is a fungal polyketide featuring an alkylated furanone core, but the biosynthetic mechanism to furnish the intri-guing molecular skeleton has yet to be elucidated. Herein, we have identified the biosynthetic gene cluster of gregatin A (1) in Penicillium sp. sh18, and investigated the mechanism that produces the intriguing structure of 1 by in vivo and in vitro recon-stitution of its biosynthesis. Our study established the biosynthetic route leading to 1, and illuminated that 1 is generated by the fusion of two different polyketide chains, which are, amazingly, synthesized by a single PKS GrgA with the aid of a trans-acting enoylreductase GrgB. Chain fusion, as well as chain hydrolysis, is catalyzed by an alpha/beta hydrolase GrgF, hybridizing the C11 and C4 carbon chains by Claisen condensation. Finally, structural analysis and mutational experiments using GrgF provided insight into how the enzyme facilitates the unusual chain-fusing reaction. In unraveling a new biosynthetic strategy involving a bifunc-tional PKS and a polyketide fusing enzyme, our study expands our knowledge concerning fungal polyketide biosynthesis.
Title: Discovery of Pyranoviolin A and Its Biosynthetic Gene Cluster in Aspergillus violaceofuscus Wei X, Chen L, Tang JW, Matsuda Y Ref: Front Microbiol, 11:562063, 2020 : PubMed
A new polyketide-non-ribosomal peptide hybrid molecule, pyranoviolin A (1), was discovered from the genome-sequenced fungus Aspergillus violaceofuscus CBS 115571 and was characterized to be the first pyranonigrin analog harboring the C-3 methoxy group. Examination of the genome sequence of the fungus identified a putative biosynthetic gene cluster of 1, which was designated as the pyv cluster. The gene deletion experiment of the polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) hybrid gene in the cluster confirmed the involvement of the pyv cluster in the pyranoviolin A biosynthesis. Finally, a plausible biosynthetic route leading to 1 has been proposed based on the bioinformatic analysis. Our study indicates that metabolite analysis of genome-sequenced microorganisms whose metabolites have been largely unexplored facilitates the discovery of new secondary metabolites along with their biosynthetic gene clusters.
Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.
The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.
Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/alpha-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/alpha-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the alpha-ketoglutarate-binding ability.
Trt14 from Aspergillus terreus is involved in unusual skeletal reconstruction during the biosynthesis of the fungal meroterpenoid terretonin. Detailed in vitro characterization revealed that this novel multifunctional enzyme catalyzes not only the D-ring expansion via intramolecular methoxy rearrangement, but also the hydrolysis of the expanded D-ring. The X-ray crystal structures of Trt14, in complex with substrate or product, and two Trt14 homologs, AusH and PrhC from Aspergillus nidulans and Penicillium brasilianum, respectively, indicated similar overall structures to those of the NTF2-like superfamily of enzymes, despite lacking sequence and functional similarities. Moreover, we gained structural insight into the mechanism of the Trt14-catalyzed ring reconstruction from the in-crystal enzyme reaction and site-directed mutagenesis to show that this reaction involves sequential ester bond cleavage and formation. Structural comparison of Trt14 and its homologs suggests that the enzymes in this new superfamily employ similar acid-base chemistry to diversify the molecular architecture of fungal meroterpenoids.
Title: Biosynthesis of LL-Z1272beta: Discovery of a New Member of NRPS-like Enzymes for Aryl-Aldehyde Formation Li C, Matsuda Y, Gao H, Hu D, Yao XS, Abe I Ref: Chembiochem, 17:904, 2016 : PubMed
LL-Z1272beta (1) is a prenylated aryl-aldehyde produced by several fungi; it also serves as a key pathway intermediate for many fungal meroterpenoids. Despite its importance in the biosynthesis of natural products, the molecular basis for the biosynthesis of 1 has yet to be elucidated. Here we identified the biosynthetic gene cluster for 1 from Stachybotrys bisbyi PYH05-7, and elucidated the biosynthetic route to 1. The biosynthesis involves a polyketide synthase, a prenyltransferase, and a nonribosomal peptide synthetase (NRPS)-like enzyme, which is responsible for the generation of the aldehyde functionality. Interestingly, the NRPS-like enzyme only accepts the farnesylated substrate to catalyze the carboxylate reduction; this represents a new example of a substrate for adenylation domains.
Paraherquonin (1), a fungal meroterpenoid produced by Penicillium brasilianum NBRC 6234, possesses a unique, highly congested hexacyclic molecular architecture. Here we identified the biosynthetic gene cluster of 1 (the prh cluster) and elucidated the pathway up to berkeleydione (2), which serves as the key intermediate for the biosynthesis of 1 as well as many other meroterpenoids. Interestingly, the nonheme iron and alpha-ketoglutarate-dependent dioxygenase PrhA constructs the cycloheptadiene moiety to afford 2 from preaustinoid A1 (6), probably via the homoallyl-homoallyl radical rearrangement. Additionally, another fungal strain, P. brasilianum MG11, which produces acetoxydehydroaustin instead of 1, was found to have a gene cluster nearly identical to the prh cluster. The dioxygenase encoded by the cluster shares 92% sequence identity with PrhA, and also accepts 6 but produces preaustinoid A3 (17) with a spiro-lactone system, generating a diverging point for the two different meroterpenoid pathways in the same species.
To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.
Title: Uncovering the unusual D-ring construction in terretonin biosynthesis by collaboration of a multifunctional cytochrome P450 and a unique isomerase Matsuda Y, Iwabuchi T, Wakimoto T, Awakawa T, Abe I Ref: Journal of the American Chemical Society, 137:3393, 2015 : PubMed
Terretonin (1) is a fungal meroterpenoid isolated from Aspergillus terreus, and possesses a highly oxygenated and unique tetracyclic structure. Although the biosynthetic gene cluster for 1 has been identified and the biosynthesis has recently been studied by heterologous reconstitution and targeted-gene deletion experiments, the last few steps of the terretonin pathway after terrenoid (6) have yet to be elucidated. Notably, the mechanism for the D-ring expansion to afford the terretonin scaffold has been a long-standing mystery to solve. Here we report the characterization of three enzymes that convert 6 into 1, as well as the complete biosynthetic pathway of 1. In the proposed terretonin pathway, the cytochrome P450 Trt6 catalyzes three successive oxidations to transform 6 into an unstable intermediate, which then undergoes the D-ring expansion and unusual rearrangement of the methoxy group to afford the core skeleton of 1. This unprecedented rearrangement is catalyzed by a novel isomerase Trt14. Finally, the nonheme iron-dependent dioxygenase Trt7 accomplishes the last two oxidation reactions steps to complete the biosynthesis.
Title: Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid Matsuda Y, Wakimoto T, Mori T, Awakawa T, Abe I Ref: Journal of the American Chemical Society, 136:15326, 2014 : PubMed
Anditomin and its precursors, andilesins, are fungal meroterpenoids isolated from Aspergillus variecolor and have unique, highly oxygenated chemical structures with a complex bridged-ring system. Previous isotope-feeding studies revealed their origins as 3,5-dimethylorsellinic acid and farnesyl pyrophosphate and suggested the possible involvement of a Diels-Alder reaction to afford the congested bicyclo[2.2.2]octane core structure of andilesins. Here we report the first identification of the biosynthetic gene cluster of anditomin and the determination of the complete biosynthetic pathway by characterizing the functions of 12 dedicated enzymes. The anditomin pathway actually does not employ a Diels-Alder reaction, but involves the nonheme iron-dependent dioxygenase AndA to synthesize the bridged-ring by an unprecedented skeletal reconstruction. Another dioxygenase, AndF, is also responsible for the structural complexification, generating the end product anditomin by an oxidative rearrangement.
Title: Spiro-ring formation is catalyzed by a multifunctional dioxygenase in austinol biosynthesis Matsuda Y, Awakawa T, Wakimoto T, Abe I Ref: Journal of the American Chemical Society, 135:10962, 2013 : PubMed
Austinol, a fungal meroterpenoid derived from 3,5-dimethylorsellinic acid, has a unique chemical structure with a remarkable spiro-lactone ring system. Despite the recent identification of its biosynthetic gene cluster and targeted gene-deletion experiments, the process for the conversion of protoaustinoid A (2), the first tetracyclic biosynthetic intermediate, to the spiro-lactone preaustinoid A3 (7) has remained enigmatic. Here we report the mechanistic details of the enzyme-catalyzed, stereospecific spiro-lactone ring-forming reaction, which is catalyzed by a non-heme iron-dependent dioxygenase, AusE, along with two flavin monooxygenases, the 5'-hydroxylase AusB and the Baeyer-Villiger monooxygenase AusC. Remarkably, AusE is a multifunctional dioxygenase that is responsible for the iterative oxidation steps, including the oxidative spiro-ring-forming reaction, to produce the austinol scaffold.
Title: Reconstituted biosynthesis of fungal meroterpenoid andrastin A Matsuda Y, Awakawa T, Abe I Ref: Tetrahedron, 69:8199, 2013 : PubMed
Andrastins (andrastin A-D), produced by several Penicillium species, exhibit inhibitory activity against ras farnesyltransferase, suggesting that these compounds could be promising leads for antitumor agents. Although the genome sequence of Penicillium chrysogenum, an andrastin-producing species, is available, the genetic and molecular bases for the biosynthesis of andrastins have not been elucidated. Here we report the identification and characterization of the gene cluster for andrastin biosynthesis. We reconstituted the biosynthetic pathway in Aspergillus oryzae, a fungal expression host, by the co-expression of five genes, including that of a terpene cyclase, and of four genes encoding the tailoring enzymes, required for the generation of andrastins. Remarkably, we successfully obtained andrastin A, the most complex andrastin molecule, as the metabolite of nine gene products, thus confirming the potential of the fungal expression system to synthesize useful natural products
BACKGROUND: Endothelial lipase (EL) is a major determinant of high-density lipoprotein-cholesterol (HDL-C) metabolism and promotes monocytes recruitment. The local expression of EL could influence atherogenesis directly, in addition to its systemic role in HDL metabolism. The EL gene has a common 584C/T polymorphism, but it is unclear whether this polymorphism is associated with HDL-C levels or acute myocardial infarction (AMI). METHODS AND RESULTS: A case-control study of 107 AMI patients and 107 control subjects was conducted. T allele frequency was lower in the AMI group than in controls (0.18 vs 0.26, p<0.05). No significant association was found between the 584C/T polymorphism and HDL-C levels. Multivariate regression analyses showed that the association of the T allele with AMI was statistically significant and independent of other risk factors when age, sex, hypertension, hypercholesterolemia, and diabetes mellitus were included in the analyses (odds ratio (OR), 0.52; 95% confidence interval (95% CI) 0.28-0.98; p=0.04). However, when smoking status was included, the association of the T allele with AMI did not remain statistically significant (OR, 0.61; 95% CI 0.32-1.18; p=0.14). CONCLUSIONS: The 584C/T polymorphism of the EL gene was associated with AMI independently of HDL-C levels and thus may be involved in the pathogenesis of AMI.
Title: Cloning and characterization of the cpyA gene encoding intracellular carboxypeptidase from Aspergillus nidulans Ohsumi K, Matsuda Y, Nakajima H, Kitamoto K Ref: Biosci Biotechnol Biochem, 65:1175, 2001 : PubMed
Carboxypeptidase Y (CPY) has been used as a maker enzyme for investigations on intracellular transport of vacuolar proteins and on vacuolar biogenesis in Saccharomyces cerevisiae. We describe the cloning and characterization of the CPY homologue encoding gene (cpyA) from the filamentous fungus Aspergillus nidulans. The cpyA gene has one intron and encodes a protein with 552 amino acids containing a putative signal sequence and pro-sequence. The predicted CpyA protein is highly similar in sequence with carboxypeptidases from several yeast species and contains a catalytic triad (Asp-His-Ser) like that of serine carboxypeptidase. The cpyA disruptant cells showed reduced levels of intracellular carboxypeptidase. These results suggest that the cpyA gene encodes a vacuolar carboxypeptidase in A. nidulans.
Title: An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans Nagai Y, Aoki J, Sato T, Amano K, Matsuda Y, Arai H and Ref: Journal of Biological Chemistry, 274:11053, 1999 : PubMed
Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192-2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5'-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA1DeltaC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1DeltaC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1DeltaC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).
We report that there is a time-related change in the phospholipase C (PLC) activities of rat brain cytosol and membrane fractions after iv injection of a soman-like or a sarin-like organophosphorous agent (bis(isopropyl methyl)phosphonate [BIMP] and bis(pinacolyl methyl)phosphonate [BPMP]). PLCgamma was activated in the brain cytosol fraction from BPMP-injected rats. The phosphorylating activity of rat brain membrane fractions were enhanced by BPMP treatment. The brain membrane fractions from BPMP-treated rats phosphorylated several proteins, including supposedly PLCgamma in the brain cytosol fraction from control rats in vitro. These results suggest that soman and sarin may stimulate a membrane tyrosine kinase, including growth factor receptors, directly or indirectly.
One of the hydrolysis products of sarin (isopropyl methylphosphonofluoridate) was detected in formalin-fixed brain tissues of victims poisoned in the Tokyo subway terrorist attack. Part of this procedure, used for the detection of sarin hydrolysis products in erythrocytes of sarin victims, has been described previously. The test materials were four individual cerebellums, which had been stored in formalin fixative for about 2 years. Sarin-bound acetylcholinesterase (AChE) was solubilized from these cerebellums, purified by immunoaffinity chromatography, and digested with trypsin. Then the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, subjected to trimethylsilyl derivatization (TMS), and detected by gas chromatography-mass spectrometry. Peaks at m/z 225 and m/z 240, which are indicative of TMS-methylphosphonic acid, were observed within the retention time range of authentic methylphosphonic acid. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of these 4 sarin victims, probably because the isopropoxy group of isopropyl methylphosphonic acid underwent chemical hydrolysis during storage. This procedure will be useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents, such as sarin, which are easily hydrolysed. This appears to be the first time that intoxication by a nerve agent has been demonstrated by analyzing formalin-fixed brains obtained at autopsy.
A new method was developed to detect sarin hydrolysis products from erythrocytes of four victims of sarin (isopropylmethylphosphonofluoridate) poisoning resulting from the terrorist attack on the Tokyo subway. Sarin-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes of sarin victims, digested with trypsin, the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, and the digested sarin hydrolysis products were subjected to trimethylsilyl derivatization and detected by gas chromatography-mass spectrometry. Isopropylmethylphosphonic acid, which is a sarin hydrolysis product, was detected in all sarin poisoning, victims we examined and methylphosphonic acid, which is a sarin and soman hydrolysis product, was determined in all victims. Postmortem examinations revealed no macroscopic and microscopic findings specific to sarin poisoning and sarin and its hydrolysis products were almost undetectable in their blood. We think that the procedure described below will be useful for the forensic diagnosis of acute sarin poisoning.
A sarin-like organophosphorus agent, [bis(isopropyl methyl)phosphonate; BIMP], was synthesized. This agent has the same phosphonate group as sarin and also has the same anti-acetylcholinesterase activity potency as sarin. The ID50 and LD50 values of BIMP in mice after intravenous injection were 3.9 nM and 0.8 mg/kg, respectively. The AChE activities of their red blood cells and brains were dose-dependently reduced by intravenous BIMP. After preparation of experimental BIMP-exposed human red blood cells, BIMP-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, digested with trypsin, and the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl (TMS) derivatization and detected by gas chromatography-mass spectrometry. Isopropyl methylphosphonic- and methylphosphonic acids, which are the sarin hydrolysis products, were detected in experimental BIMP-exposed human red blood cells. This new method, which enables sarin's hydrolysis products to be detected in BIMP-exposed erythrocytes, is a useful tool for studying sarin-poisoning victims.
