A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.
2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter−1) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter−1) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.
Mycobacterium tuberculosis α–isopropylmalate synthase (MtIPMS) catalyzes the condensation of AcCoA with α–ketoisovalerate (α–KIV) and the subsequent hydrolysis of α–isopropylmalyl-CoA to generate the products CoA and α–isopropylmalate (α–IPM). This is the first committed step in L–leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and 1H-NMR spectroscopy. The α–keto acid specificity of the enzyme is narrow and the acyl-CoA specificity is absolute for AcCoA. In the absence of α–KIV MtIPMS does not enolize the α–protons of AcCoA, but slowly hydrolyzes acyl-CoA analogs. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random Bi Bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pKa values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of α–isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate limiting, although with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of α–isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. Based on these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.
α-Isopropylmalate Synthase; Steady-State Kinetics; Chemical Mechanism
d-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 μmol/min mg (protein)−1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes an operon. By using panC, an assay system was developed to quantify d-pantothenate. The wild type of C. glutamicum was found to accumulate 9 μg of this vitamin per liter. A strain was constructed (i) to abolish l-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to d-pantothenate. The best resulting strain has ilvA deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (ilvBNCD) and d-pantothenate (panBC) synthesis. With this strain a d-pantothenate accumulation of up to 1 g/liter is achieved, which is a 105-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the d-pantothenate-specific part of the pathway and that the availability of β-alanine is essential for d-pantothenate formation.
Beauvericin is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and which can induce apoptosis in mammalian cells. Beauvericin is produced by some entomo- and phytopathogenic Fusarium species (Fusarium proliferatum, F. semitectum, and F. subglutinans) and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. We tested 94 Fusarium isolates belonging to 25 taxa, 21 in 6 of the 12 sections of the Fusarium genus and 4 that have been described recently, for the ability to produce beauvericin. Beauvericin was produced by the following species (with the number of toxigenic strains compared with the number of tested strains given in parentheses): Fusarium acuminatum var. acuminatum (1 of 4), Fusarium acuminatum var. armeniacum (1 of 3), F. anthophilum (1 of 2), F. avenaceum (1 of 6), F. beomiforme (1 of 1), F. dlamini (2 of 2), F. equiseti (2 of 3), F. longipes (1 of 2), F. nygamai (2 of 2), F. oxysporum (4 of 7), F. poae (4 of 4), F. sambucinum (12 of 14), and F. subglutinans (3 of 3). These results indicate that beauvericin is produced by many species in the genus Fusarium and that it may be a contaminant of cereals other than maize.
Escherichia coli can synthesize alpha-ketoisovalerate, the precursor of valine, leucine, and pantothenate, by three routes: anabolically via dihydroxyacid dehydrase and catabolically via both the branched-chain amino acid transaminase (transaminase B) and the alanine-valine transaminase (transaminase C). An E. coli K-12 mutant devoid of transaminase C (avtA) was isolated by mutagenizing an isoleucine-requiring strain devoid of transaminase B (ilvE::Tn5) with Mu d1(Ap lac) and selecting for valine-requiring derivatives which were ampicillin resistant, Lac+, able to crossfeed an ilvD mutant, and unable to grow on alpha-ketoisovalerate in place of valine. Strains defective in one, two, or all three alpha-ketoisovalerate metabolic enzymes were constructed, and their properties were analyzed. The data indicated that avtA is the structural gene for transaminase C, that transaminase C is a single enzyme species, and that the sole pathway for pantothenate biosynthesis is from alpha-ketoisovalerate. The data further showed that isoelectric inhibits the transaminase B-catalyzed deamination of valine in vivo.
We have cloned the cDNA encoding the KIV chain of a human antibody with specificity against the major carbohydrate antigen of Streptococcus A. The cDNA has been used as a genetic probe to estimate the number of germline VKIV genes in human DNA. The presence of unique hybridizing bands on digestion of human DNA with several restriction endonucleases and the equivalence of the DNA in a band to a single gene per haploid genome point to the conclusion that there is a unique human VKIV germline gene. The corollary of this conclusion is that the diversity of human VKIV chains must be exclusively due to somatic mutation. This is supported by examination of the sequences of human KIV chain genes and their KIV chain products. Fusion of the unique germline VKIV gene (1) with one of several JK segments, followed by somatic mutations in the V region of the rearranged KIV gene, can account for the known sequences. The restricted germline gene repertoire may account for the small proportion of human KIV chains in the human K chain sequence library (2).
Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.
Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.
Background and Purpose
Lipoprotein(a) level (Lp(a)) is an established risk factor for coronary artery disease and has been implicated in carotid artery disease (CAAD). The relationship between genetic variation in the LPA gene region and CAAD risk remains unknown.
We genotyped single nucleotide polymorphisms (SNPs) in the LPAL2, LPA, and PLG region in 530 individuals with severe CAAD and 770 controls and kringle IV type 2 (KIV2) repeat length in a subset of 90 individuals.
Nine SNPs collectively accounted for 30% of the variance in Lp(a) level. Six SNPs were associated with Lp(a) level after accounting for KIV2 copy number, and the dominant KIV2 allele combined with these markers explained 60% of the variance in Lp(a) level. Five SNPs, including rs10455872, which had an odds ratio of 2.1 per minor allele, and haplotypes formed by rs10455872, rs6919346, and rs3123629 were significant predictors of CAAD. After accounting for Lp(a) level, all evidence of CAAD-genotype association in the LPA region was eliminated.
LPA region SNPs capture some but not all of the effect of KIV2 repeat length on Lp(a) level. There are associations between LPA region SNPs and CAAD which appear to be due to effects on Lp(a) level.
Carotid stenosis; atherosclerosis; lipoprotein(a); genomics; risk factors
Beauvericin, a cyclodepsipeptide, was produced by cultures of three strains of Fusarium proliferatum, M-5991, M-6992, and M-6993, grown on cracked corn. M-5991 produced approximately 1,000-mg/kg levels of fumonisins, moniliformin, and beauvericin.
Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected.
Trichoderma reesei; Aspergillus niger; Fusarium graminearum; Erythrose reductase; Erythritol
For two fungal strains to be vegetatively compatible and capable of forming a stable vegetative heterokaryon they must carry matching alleles at a series of loci variously termed het or vic genes. Cloned het/vic genes from Neurospora crassa and Podospora anserina have no obvious functional similarity and have various cellular functions. Our objective was to identify the homologue of the Neurospora het-c gene in Fusarium proliferatum and to determine if this gene has a vegetative compatibility function in this economically important and widely dispersed fungal pathogen. In F. proliferatum and five other closely related Fusarium species we found a few differences in the DNA sequence, but the changes were silent and did not alter the amino acid sequence of the resulting protein. Deleting the gene altered sexual fertility as the female parent, but it did not alter male fertility or existing vegetative compatibility interactions. Replacement of the allele-specific portion of the coding sequence with the sequence of an alternate allele in N. crassa did not result in a vegetative incompatibility response in transformed strains of F. proliferatum. Thus, the fphch gene in Fusarium appears unlikely to have the vegetative compatibility function associated with its homologue in N. crassa. These results suggest that the vegetative compatibility phenotype may result from convergent evolution. Thus, the genes involved in this process may need to be identified at the species level or at the level of a group of species and could prove to be attractive targets for the development of antifungal agents.
Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B1 was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.
Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B1–B3, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB1 content was 250 μg g−1 in pineapple skin and 20 μg ml−1 in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.
Electronic supplementary material
The online version of this article (doi:10.1007/s13353-013-0146-0) contains supplementary material, which is available to authorized users.
Ananas comosus; FUM genes; Mycotoxins; Phylogeny; Tropical fruit diseases
HSAF was isolated from Lysobacter enzymogenes, a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here, we described the HSAF biosynthetic gene cluster which contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes, encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activated L-ornithine and the four-domain NRPS was able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.
Nonribosomal peptide synthetases (NRPSs) are multienzymatic, multidomain megasynthases involved in the biosynthesis of pharmaceutically important nonribosomal peptides. The peptaibol synthetase from Trichoderma virens (TPS) is an important member of the NRPS family that exhibits antifungal properties. The majority of the NRPSs terminate peptide synthesis with the thioesterase (TE) domain, which either hydrolyzes the thioester linkage, releasing the free peptic acid, or catalyzes the intramolecular macrocyclization to produce a macrolactone product. TPS is an important NRPS that does not encompass a TE domain, but rather a reductase domain (R domain) to release the mature peptide product reductively with the aid of a NADPH cofactor. However, the catalytic mechanism of the reductase domain has not yet been elucidated.
We present here a three-dimensional (3D) model of the reductase domain based on the crystal structure of vestitone reductase (VR). VR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and is responsible for the nicotinamide dinucleotide phosphate (NADPH)-dependent reduction of the substrate into its corresponding secondary alcohol product. The binding sites of the probable linear substrates, alamethicin, trichotoxin, antiamoebin I, chrysopermin C and gramicidin, were identified within the modeled R domain using multiple docking approaches. The docking results of the ligand in the active site of the R domain showed that reductase side chains have a high affinity towards ligand binding, while the thioester oxygen of each substrate forms a hydrogen bond with the OH group of Tyr176 and the thiol group of the substrate is closer to the Glu220. The modeling and docking studies revealed the reaction mechanism of reduction of thioester into a primary alcohol.
Peptaibol biosynthesis incorporates a single R domain, which appears to catalyze the four-electron reduction reaction of a peptidyl carrier protein (PCP)-bound peptide to its corresponding primary alcohol. Analysis of R domains present in the non-redundant (nr) database of the NCBI showed that the R domain always resides in the last NRPS module and is involved in either a two or four-electron reduction reaction.
Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5′ noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.
Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.
The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.
Tolypocladium inflatum, the fungus from which the immunosuppressant drug cyclosporin was isolated, is a prolific producer of secondary metabolites with potential applications in medicine and agriculture. We have sequenced the first draft reference genome of T. inflatum, which also represents the first genome of a novel family of insect pathogenic fungi, Ophiocordycipitaceae. We present comparative genomic and evolutionary analyses of the cyclosporin nonribosomal peptide synthetase (simA), which highlight the lineage specific nature of cyclosporin's origin and the homology of cyclosporin adenylation (A) domains with other fungal NRPSs whose products show anti-insect activity. RNA-Seq data profiles the expression patterns of the cyclosporin gene cluster in an inducing medium and in response to media simulating distinct stages of insect pathogenesis. Sequencing of the T. inflatum genome has uncovered the metabolite gene cluster responsible for cyclosporin biosynthesis and characterized complex patterns of its evolution.
The metabolic pathways utilized by an obligately anaerobic marine spirochete (strain MA-2) to ferment branched-chain amino acids were studied. The spirochete catabolized l-leucine to isovaleric acid, l-isoleucine to 2-methylbutyric acid, and l-valine to isobutyric acid, with accompanying CO2 production in each fermentation. Cell extracts of spirochete MA-2 converted l-leucine, l-isoleucine, and l-valine to 2-ketoisocaproic, 2-keto-3-methylvaleric, and 2-ketoisovaleric acids, respectively, through mediation of 2-ketoglutarate-dependent aminotransferase activities. The branched-chain keto acids were decarboxylated and oxidized to form isovaleryl coenzyme A, 2-methylbutyryl coenzyme A, and isobutyryl coenzyme A, respectively, in the presence of sulfhydryl coenzyme A and benzyl viologen. The acyl coenzyme A's were converted to acyl phosphates by phosphate branched-chain acyltransferase enzymatic activities. Branched-chain fatty acid kinase activities catalyzed formation of isovaleric, 2-methylbutyric, and isobutyric acids from isovaleryl phosphate, 2-methylbutyryl phosphate, and isobutyryl phosphate, respectively. Adenosine 5′-triphosphate was formed during conversion of branched-chain acyl phosphates to branched-chain fatty acids. The results indicate that conversion of l-leucine, l-isoleucine, and l-valine to branched-chain fatty acids by spirochete MA-2 results in adenosine 5′-triphosphate generation. The metabolic pathways utilized for this conversion involve amino acid amino-transferase, 2-keto acid oxidoreductase, phosphate acyltransferase, and fatty acid kinase activities.
Cell-free extracts of Acetobacter suboxydans were prepared which were capable of condensing α-ketoisovalerate with 14C-labeled acetyl-coenzyme A to yield 14C-labeled α-isopropylmalate. The product of the reaction was isolated by paper and column chromatography and was characterized by recrystallization with synthetic α-isopropylmalic acid to constant specific radioactivity. The formation of α-isopropylmalate by extracts of A. suboxydans plus the ability of the organism to grow in a simple glucose-glycerol medium containing glutamic acid as the only amino acid indicate that the pathway for leucine biosynthesis shown to exist in yeast and Salmonella typhimurium also occurs in A. suboxydans. As a comparison, the condensation of oxalacetate and (14C) acetyl-coenzyme A to yield (14C) citric acid was shown, by similar means, to occur in A. suboxydans. This is of interest since the existence of this classical condensing enzyme has hitherto not been demonstrated in this organism. This reaction was further demonstrated in cell-free extracts of A. suboxydans by means of a spectrophotometric assay at 232 mμ which measured the cleavage of the carbon-sulfur bond of acetyl-coenzyme A in the presence of oxalacetate. Comparison of the specific activities of crude cell-free extracts indicated a much more extensive occurrence of this reaction in yeast than in A. suboxydans.
Deuterated type III antifreeze protein specifically hydrogen reverse-labelled in the methyl groups of leucine and valine residues has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.80 Å resolution from a 0.23 mm3 crystal.
Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor α-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D2O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 Å from a crystal of dimensions 1.60 × 0.38 × 0.38 mm corresponding to a volume of 0.23 mm3.
ocean pout type III antifreeze protein; neutron diffraction; reverse labelling; deuteration
Salmonella typhimurium strain DU501, which was found to be deficient in acetohydroxy acid synthase II (AHAS II) and to possess elevated levels of transaminase B and biosynthetic threonine deaminase, required isoleucine, methionine, or pantothenate for growth. This strain accumulated α-ketobutyrate and, to a lesser extent, α-aminobutyrate. We found that α-ketobutyrate was a competitive substrate for ketopantoate hydroxymethyltransferase, the first enzyme in pantothenate biosynthesis. This competition with the normal substrate, α-ketoisovalerate, limited the supply of pantothenate, which resulted in a requirement for methionine. Evidence is presented to support the conclusion that the ambivalent requirement for either pantothenate or methionine is related to a decrease in succinyl coenzyme A, which is produced from pantothenate and which is an obligatory precursor of methionine biosynthesis. The autointoxification by endogenously produced α-ketobutyrate could be mimicked in wild-type S. typhimurium by exogenously supplied α-ketobutyrate or salicylate, a known inhibitor of pantothenate biosynthesis. The accumulation of α-ketobutyrate was initiated by the inability of the residual AHAS activity provided by AHAS I to efficiently remove the α-ketobutyrate produced by biosynthetic threonine deaminase. The accumulation of α-ketobutyrate was amplified by the action of transaminase B, which decreased the isoleucine pool by catalyzing the formation of α-keto-β-methylvalerate and aminobutyrate from isoleucine and α-ketobutyrate; this resulted in release of threonine deaminase from end product inhibition and unbridled production of α-ketobutyrate. Isoleucine satisfied the auxotrophic requirement of the AHAS II-deficient strain by curtailing the activity of threonine deaminase. Additional lines of evidence based on genetic and physiological experiments are presented to support the basis for the autointoxification of strain DU501 as well as other nonpolarigenic ilvG mutant strains.
Three adult human subjects were maintained for 7 days (period I) on a protein-free formula diet containing the minimum daily requirements of the eight essential amino acids plus 40 g glycine. During the last 5 days of this period, the average daily nitrogen balances for the three subjects were +0.52, +0.71, and +0.30 g, respectively. During the next 7 days (period II), valine was withdrawn from the diet, and the glycine ration increased by an equimolar amount. During the last 5 days of period II, average daily nitrogen balances declined to -1.82, -1.61, and -1.87 g, respectively. In the final period of 7 days (period III), the keto analogue of valine, α-ketoisovaleric acid, was added to the diet in a quantity equimolar to the minimum daily requirement of valine. During the last 5 days of this period, average daily nitrogen balances improved to -0.02, -0.18, and -0.83 g, respectively. Analogous experiments in three subjects involved the withdrawal from the diet of phenylalanine (period II) and replacement by its keto analogue, phenylpyruvic acid (period III). The average daily nitrogen balances were as follows: period I: +1.04, +0.96, and +0.53 g; period II: -1.45, -1.83, and -1.94 g; period III: +0.07, +0.11, and -0.52 g.
The data demonstrate that man can convert α-ketoisovaleric acid and phenylpyruvic acid to the corresponding essential amino acids, valine and phenylalanine. The efficiency of these conversions is considerably less than 100%.