Expression of the trichodiene synthase gene of Fusarium sporotrichioides in Escherichia coli results in sesquiterpene production

Trichodiene synthase is a sesquiterpene cyclase involved in the biosynthesis of trichothecene mycotoxins. We report that insertion of the unaltered trichodiene synthase gene of Fusarium sporotrichioides into the Escherichia coli expression vector pDR540 produced an inactive polypeptide with a molecular weight approximately 2000 greater than that of trichodiene synthase. This result is consistent with the presence of an intron in the trichodiene synthase gene, and prompted us to specifically delete a putative 60-nucleotide intron sequence. Insertion of the intron-deleted open reading frame into pDR540 resulted in the production of active enzyme. Trichodiene synthase activity in crude extracts from induced cultures was 0.07 nmol/min/mg of protein and represented 0.05-0.10% of the total cell protein. A cross-reactive protein was present with the same apparent molecular weight as the subunit of native trichodiene synthase. The recombinant enzyme was partially purified and shown to have properties closely resembling those of the native enzyme. Trichodiene was detected in ethyl acetate extracts from induced cultures at a concentration of 60 micrograms/liter after 4.5 h. These findings support the primary structure recently reported for trichodiene synthase and demonstrate that the expression of a sesquiterpene cyclase in E. coli results in sesquiterpene production.     

Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides

The trichodiene synthase gene (Tox5) has been isolated from the fungus Fusarium sporotrichioides, and its nucleotide (nt) sequence determined. A lambda gt11 library of F. sporotrichioides DNA was screened with antiserum against trichodiene synthase (TS). DNA fragments were isolated which encode a portion of the Tox5 gene. In subsequent screening of the library we employed one of these DNAs as a probe and identified several recombinant phage containing the entire Tox5 gene. The gene consists of a 1182-nt open reading frame (ORF) which contains a 60-nt intron and specifies a Mr 43,999 protein. The deduced amino acid sequence of the ORF was identical to sequences determined for several CNBr peptides from purified TS. Southern and Northern analyses indicated that the Tox5 gene is present in a single copy and is transcribed into an mRNA of about 1450 nt. Upstream from the start codon, 'TATA'-like sequences and a short repeated sequence resembling the 'CCAAT' box were observed. The primary structure described for TS is the first such report for a member of the terpene cyclase group of enzymes.     

Purification and characterization of the sesquiterpene cyclase patchoulol synthase from Pogostemon cablin

The sesquiterpene cyclase, patchoulol synthase, from Pogostemon cablin (patchouli) leaves was purified to apparent homogeneity by chromatofocusing, anion exchange, gel permeation, and hydroxylapatite chromatography. The enzyme showed a maximum specific activity of about 20 nmol/min/mg protein, and a native molecular weight of 80,000 as determined by gel permeation chromatography. The protein was very hydrophobic, as judged by chromatographic behavior on several matrices, and possessed a pI value of about 5.0, as determined by isoelectric and chromatofocusing. SDS-PAGE showed the enzyme to be composed of two apparently identical subunits of Mr approximately 40,000. Maximum activity was observed at pH 6.7 in the presence of Mg2+ (Km approximately 1.7 mM); other divalent metal ions were ineffective in promoting catalysis. The Km value for the substrate, farnesyl pyrophosphate, was 6.8 microM. Patchoulol synthase copurified with the ability to transform farnesyl pyrophosphate to cyclic olefins (alpha- and beta-patchoulene, alpha-bulnesene, and alpha-guiaene) and this observation, plus evidence based on differential inhibition and inactivation studies, suggested that these structurally related products are synthesized by the same cyclase enzyme. In general properties, the patchoulol synthase from patchouli leaves resembles fungal sesquiterpene olefin cyclases except for the ability to synthesize multiple products, a property more typical of monoterpene cyclases of higher plant origin.     

Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures

An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a K_m for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.     

Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene in Fusarium sporotrichioides and Gibberella pulicaris

Ancymidol, a plant growth regulator, inhibited biosynthesis of diacetoxyscirpenol by Gibberella pulicaris (Fusarium sambucinum) in a defined liquid medium. Ancymidol also inhibited biosynthesis of T-2 toxin by a wild-type strain of Fusarium sporotrichioides and biosynthesis of diacetoxyscirpenol, deacetylated calonectrin, and dideacetylated calonectrin by mutant strains of this species. Ancymidol-treated cultures accumulated the hydrocarbon trichodiene, a biosynthetic precursor of the trichothecenes. Ancymidol did not block trichodiene accumulation by a trichodiene-producing mutant strain of F. sporotrichioides. Ancymidol appears to block the trichothecene biosynthetic pathway after formation of trichodiene and before formation of trichothecenes containing four or more oxygen atoms.     

Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene in Fusarium sporotrichioides and Gibberella pulicaris.

Ancymidol, a plant growth regulator, inhibited biosynthesis of diacetoxyscirpenol by Gibberella pulicaris (Fusarium sambucinum) in a defined liquid medium. Ancymidol also inhibited biosynthesis of T-2 toxin by a wild-type strain of Fusarium sporotrichioides and biosynthesis of diacetoxyscirpenol, deacetylated calonectrin, and dideacetylated calonectrin by mutant strains of this species. Ancymidol-treated cultures accumulated the hydrocarbon trichodiene, a biosynthetic precursor of the trichothecenes. Ancymidol did not block trichodiene accumulation by a trichodiene-producing mutant strain of F. sporotrichioides. Ancymidol appears to block the trichothecene biosynthetic pathway after formation of trichodiene and before formation of trichothecenes containing four or more oxygen atoms.     

Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis

Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism.     

Purification and characterization of ADP-ribosyl cyclase from Euglena gracilis

ADP-ribosyl cyclase, which catalyzes the conversion from NAD+ to cyclic adenosine diphosphoribose (cADPR), is proposed to participate in cell cycle regulation in Euglena gracilis. This enzyme, which was found as a membrane-bound protein, was purified almost the homogeneity after solubilization with deoxycholate, and found to be a monomeric protein with a molecular mass of 40 kDa. Its Km value for NAD+ was estimated to be 0.4 mM, and cADPR, a product of the enzyme, inhibited the enzyme competitively with respect to NAD+ whereas another product, nicotinamide, showed noncompetitive (mixed-type) inhibition. In contrast to mammalian CD38 and BST-1, Euglena ADP-ribosyl cyclase lacked cADPR hydrolase activity.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes

The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence.     

Purification and characterization of glutamine synthetase from Rhodomicrobium vannielii

Abstract Glutamine synthetase (GS) from the purple non-sulfur bacterium Rhodomicrobium vannielii has been purified to electrophoretic homogeneity by affinity chromatography. Molecular weight and catalytic properties of the enzy,e are similar to those described for other species of Rhodospirillaceae. However, the enzyme from this organism appears to be antigenically different from the glutamine synthetases of other species of Rhodospirillaceae studied.     

Structural and functional characterization of the TRI101 trichothecene 3-O-acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum: kinetic insights to combating Fusarium head blight

Fusarium head blight (FHB) is a plant disease with serious economic and health impacts. It is caused by fungal species belonging to the genus Fusarium and the mycotoxins they produce. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by approximately 100-fold but has demonstrated limited resistance to FHB in transgenic cereal. To understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes toward B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three-dimensional structures for the ternary complexes for both of these enzymes with coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein.     

Purification and characterization of guanylate cyclase from Caulobacter crescentus

Guanylate cyclase has been purified 60-fold from cell extracts of the bacterium $\text{Caulobacter crescentus}$. It has a molecular weight of approximately 140,000 and is dependent upon Mn²⁺ for activity. Enzymic activity is unaffected by cyclic AMP, cyclic GMP or N⁶,O^2′-dibutyryl cyclic AMP but is stimulated by N²,O^2′-dibutyryl cyclic GMP. The partially purified preparation of guanylate cyclase does not contain detectable adenylate cyclase activity.     

Purification and characterization of invertase isozymes from Fusarium oxysporum

Fusarium invertase appears to exist in two forms, mycelial (P-1) and conidial (P-2) types. They were purified and partially characterized, but their specific activities were too low when compared with yeast and Neurospora invertases. Present studies describe a method for isolation of highly purified enzymes and their properties. The enzymes are homogeneous by several criteria. Estimation of molecular weights revealed the subunit structure of each invertase, and the association-dissociation of subunits seem to occur as temperature varies. Amino acid compositions and other properties have been studied. The two invertases are glycoproteins which contain 36% (P-1) and 23% (P-2) carbohydrates (predominantly mannose with smaller percentages of glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine). Comparison with the properties of the previous preparations is also described.     

Isolation and characterization of two new trichothecenes from Fusarium sporotrichioides strain M-1-1.

Two new trichothecenes were isolated along with T-2 toxin, neosolaniol, and HT-2 toxin from the culture filtrate of Fusarium sporotrichioides strain M-1-1. The structures of the new toxins were characterized to be 4 beta, 8 alpha-diacetoxy-12,13-epoxytrichothec-9-ene-3 alpha, 15-diol (designated NT-1) and 4 beta-acetoxy-12,13-epoxy-trichothec-9-ene-3 alpha,8 alpha,15-triol (designated NT-2).     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum.

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization

S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.