Involvement of two cytosolic enzymes and a novel intermediate, 5'-oxoaverantin, in the pathway from 5'-hydroxyaverantin to averufin in aflatoxin biosynthesis

During aflatoxin biosynthesis, 5'-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5'-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.     

Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′S,5′S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5′-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.     

adhA in Aspergillus parasiticus Is Involved in Conversion of 5′-Hydroxyaverantin to Averufin

Two routes for the conversion of 5′-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5′-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA, from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases—a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD⁺-NADP⁺, and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA (adhA1), whereas A. parasiticus SRRC 2043, a strain that accumulates O-methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.     

Aspergillus parasiticus cyclase catalyzes two dehydration steps in aflatoxin biosynthesis

In the aflatoxin biosynthetic pathway, 5'-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2'S,5'S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5'-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.     

Purification and Gene Cloning of a Dehydrogenase from Lactobacillus brevis That Catalyzes a Reaction Involved in Aflatoxin Biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5′-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5′-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS–PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.     

Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.     

Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved in aflatoxin biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5'-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5'-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.     

Enzymatic Conversion of Averufin to Hydroxyversicolorone and Elucidation of a Novel Metabolic Grid Involved in Aflatoxin Biosynthesis

The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus. When (1′S,5′S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1′R,5′R)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with A. parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1′S,5′S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.     

Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis

5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).     

Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis.

5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).     

Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R² = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Formation of adenosine 5'-phosphorofluoridate and the assay of adenyl cyclase in barley seeds

Barley seed (Hordeum vulgare L.) homogenates contain an apparent enzymatic activity which catalyzes the synthesis of adenosine 5′-phosphorofluoridate from magnesium-adenosine 5′-triphosphate and sodium fluoride. Formation of this compound may interfere with some adenyl cyclase assays which use fluoride as a component of the incubation medium. Neither adenyl cyclase activity nor endogenous adenosine 3′: 5′-monophosphate was detected in barley seed homogenates or extracts.     

Function of the cypX and moxY genes in aflatoxin biosynthesis in Aspergillus parasiticus

The pathway oxoaverantin (OAVN) --> averufin (AVR) --> hydroxyversicolorone (HVN) --> versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.     

Purification and characterization of a new NAD(+)-dependent enzyme, L-tartrate decarboxylase, from Pseudomonas sp. group Ve-2.

A new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarboxylation on NAD+ suggests that the decarboxylation involves redox reactions of the substrate. The enzyme catalyzes NAD(+)-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40,000). The apparent Michaelis constants for L-tartrate and NAD+ are 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomonas sphaeroides, and tartrate dehydrogenase from P. putida.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

Stereochemistry during aflatoxin biosynthesis: conversion of norsolorinic acid to averufin.

A reaction sequence, norsolorinic acid (NA)-->averantin (AVN)-->5'-hydroxyaverantin (HAVN)-->averufin (AVR), is the early part of a biosynthetic pathway for aflatoxins. In this study, we determined the stereochemical relationship among these metabolites by using chiral high-performance liquid chromatography. In cell-free experiments using the cytosol fraction of Aspergillus parasiticus NIAH-26, (1'S)-AVN was exclusively produced from NA in the presence of NADPH. Also, only (1'S)-AVN, and not (1'R)-AVN, served as a substrate for the reverse reaction from AVN to NA. When the microsome fraction of NIAH-26 was incubated with (1'S)-AVN in the presence of NADPH, two HAVN diastereomers and one AVR enantiomer were formed, whereas these substances were never produced from (1'R)-AVN. Moreover, (1'S,5'R)-AVR was exclusively formed from both HAVN diastereomers by the cytosol fraction in the presence of NAD. The feeding experiments using this mutant showed that aflatoxins were produced from (1'S,5'R)-AVR but not from (1'R,5'S)-AVR. These results indicate that the enzymes involved in this pathway show strict stereospecificity to their substrates and that the configuration of (1'S,5'R)-AVR leading to the formation of aflatoxins is due to the stereospecificity of NA dehydrogenase which catalyzes the reaction between (1'S)-AVN and NA.     

Biochemical and Genetic Characterization of trans-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading Nocardioides Strain

trans-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The K_m and kcat values of this enzyme for trans-2′-carboxybenzalpyruvate were 50 μM and 13 s⁻¹, respectively. trans-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2′-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.     

Identification and characterization of a short 2′–3′ bond-forming ribozyme

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2′–3′ phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.     

RNA 5′-Triphosphatase, Nucleoside Triphosphatase, and Guanylyltransferase Activities of Baculovirus LEF-4 Protein

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5′-triphosphatase that hydrolyzes the γ phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and P_i (K_m = 43 μM ATP; V_max = 30 s⁻¹) and GTP to GDP and P_i. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.     

Participation in aflatoxin biosynthesis by a reductase enzyme encoded by vrdA gene outside the aflatoxin gene cluster

Three reactions from hydroxyversicolorone to versicolorone, from versiconal hemiacetal acetate to versiconol acetate, and from versiconal to versiconol are involved in a metabolic grid in aflatoxin biosynthesis. This work demonstrated that the same reductase of Aspergillus parasiticus catalyzes the three reactions. The gene (named vrdA) encoding the reductase was cloned, and its sequence did not show homology to any regions in aflatoxin gene cluster. Its cDNA encoding a 38,566 Da protein was separated by three introns in the genome. Deletion of the vrdA gene in A. parasiticus caused a significant decrease in enzyme activity, but did not affect aflatoxin productivity of the fungi. Although the vrdA gene was expressed in culture conditions conducive to aflatoxin production, it was expressed even in the aflR deletion mutant. These results suggest that the vrdA is not an aflatoxin biosynthesis gene, although it actually participates in aflatoxin biosynthesis in cells.