Multienzyme Process Research Articles

Complex Interaction between Iron, Vitamin B2, Vitamin B12 and Vitamin D During the Activation of Vitamin D

Functional Vitamin D is required for absorption of calcium from the intestine and for regulating calcium and phosphorus deposition to ensure healthy bone density. More recently it has been found that vitamin D is also essential for maturation of neuronal stem cells. Activation of vitamin D is a multi-enzyme process, which requires a contribution of several co-factors including iron, vitamin B2 and vitamin B12. We have used urinary phosphoric acid as a marker of functional vitamin D deficiency, and have compared various urinary metabolic markers with phosphoric acid levels to follow the essential elements in vitamin D activation. Levels of phosphoric acid were increased in each of iron, vitamin B2 and vitamin B12 deficiency, with the greatest increase seen in low functional vitamin B12.

High-Yield Synthesis of Enantiopure 1,2-Amino Alcohols from l-Phenylalanine via Linear and Divergent Enzymatic Cascades.

Enantiomerically pure 1,2-amino alcohols are important compounds due to their biological activities and wide applications in chemical synthesis. In this work, we present two multienzyme pathways for the conversion of l-phenylalanine into either 2-phenylglycinol or phenylethanolamine in the enantiomerically pure form. Both pathways start with the two-pot sequential four-step conversion of l-phenylalanine into styrene via subsequent deamination, decarboxylation, enantioselective epoxidation, and enantioselective hydrolysis. For instance, after optimization, the multienzyme process could convert 507 mg of l-phenylalanine into (R)-1-phenyl-1,2-diol in an overall isolated yield of 75% and >99% ee. The opposite enantiomer, (S)-1-phenyl-1,2-diol, was also obtained in a 70% yield and 98–99% ee following the same approach. At this stage, two divergent routes were developed to convert the chiral diols into either 2-phenylglycinol or phenylethanolamine. The former route consisted of a one-pot concurrent interconnected two-step cascade in which the diol intermediate was oxidized to 2-hydroxy-acetophenone by an alcohol dehydrogenase and then aminated by a transaminase to give enantiomerically pure 2-phenylglycinol. Notably, the addition of an alanine dehydrogenase enabled the connection of the two steps and made the overall process redox-self-sufficient. Thus, (S)-phenylglycinol was isolated in an 81% yield and >99.4% ee starting from ca. 100 mg of the diol intermediate. The second route consisted of a one-pot concurrent two-step cascade in which the oxidative and reductive steps were not interconnected. In this case, the diol intermediate was oxidized to either (S)- or (R)-2-hydroxy-2-phenylacetaldehyde by an alcohol oxidase and then aminated by an amine dehydrogenase to give the enantiomerically pure phenylethanolamine. The addition of a formate dehydrogenase and sodium formate was required to provide the reducing equivalents for the reductive amination step. Thus, (R)-phenylethanolamine was isolated in a 92% yield and >99.9% ee starting from ca. 100 mg of the diol intermediate. In summary, l-phenylalanine was converted into enantiomerically pure 2-phenylglycinol and phenylethanolamine in overall yields of 61% and 69%, respectively. This work exemplifies how linear and divergent enzyme cascades can enable the synthesis of high-value chiral molecules such as amino alcohols from a renewable material such as l-phenylalanine with high atom economy and improved sustainability.

Role of the Ubiquitin Proteasome System in the Regulation of Blood Pressure: A Review.

The kidney and the vasculature play crucial roles in regulating blood pressure. The ubiquitin proteasome system (UPS), a multienzyme process mediating covalent conjugation of the 76-amino acid polypeptide ubiquitin to a substrate protein followed by proteasomal degradation, is involved in multiple cellular processes by regulating protein turnover in various tissues. Increasing evidence demonstrates the roles of UPS in blood pressure regulation. In the kidney, filtered sodium is reabsorbed through diverse sodium transporters and channels along renal tubules, and studies conducted till date have provided insights into the complex molecular network through which ubiquitin ligases modulate sodium transport in different segments. Components of these pathways include ubiquitin ligase neuronal precursor cell-expressed developmentally downregulated 4-2, Cullin-3, and Kelch-like 3. Moreover, accumulating data indicate the roles of UPS in blood vessels, where it modulates nitric oxide bioavailability and vasoconstriction. Cullin-3 not only regulates renal salt reabsorption but also controls vascular tone using different adaptor proteins that target distinct substrates in vascular smooth muscle cells. In endothelial cells, UPS can also contribute to blood pressure regulation by modulating endothelial nitric oxide synthase. In this review, we summarize current knowledge regarding the role of UPS in blood pressure regulation, focusing on renal sodium reabsorption and vascular function.

Polysaccharide Sequence Influences the Specificity and Catalytic Activity of Glucuronyl C5-Epimerase.

Heparin is a widely used biotherapeutic produced from animal tissues. However, it might be possible to produce a bioengineered version using a multienzyme process, relying on the isolation of the E. coli K5 capsule heparosan and its chemical conversion to N-sulfoheparosan, NSH. Glucuronyl C5-epimerase, the first enzyme that acts on NSH, catalyzes the reversible conversion of glucuronic acid (GlcA) to iduronic acid (IdoA). Using full-length NSH, containing different amounts of N-acetylglucosamine (GlcNAc) residues, we demonstrate that C5-epimerase specificity relates to polysaccharide sequence, particularly the location of GlcNAc residues within the chain. We leveraged the deuterium exchange and the novel β-glucuronidase heparanase BP, which cleaves at the GlcA residue. Liquid chromatography-mass spectrometry and gel permeation chromatography of partial/complete heparanase BP digestion products from various NSH substrates treated with C5-epimerase provide information on C5-epimerase activity and action pattern. This study provides insight into optimizing the large-scale production of bioengineered heparin.

Elucidating the unusual reaction kinetics of D-glucuronyl C5-epimerase.

The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3'-phosphoadenosine 5'-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis-Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.

Ammonium Gluconate Production from Potato Starch Wastewater Using a Multi-Enzyme Process

In China, potato starch wastewater causes serious environmental pollution. Therefore, in this study, we aimed to develop a method for the transformation of potato starch in potato starch wastewater into ammonium gluconate, where potato starch wastewater was enzymatically hydrolyzed by liquefaction and saccharification. The optimum parameters of the hydrolysis process were as follows: α-amylase concentration, 0.5% (v/v); reaction time, 35 min; temperature, 90 °C; glucoamylase concentration, 0.7% (v/v); reaction time, 25 h; temperature, 50 °C; and pH, 4.0. Subsequently, ammonium gluconate was produced using 4.6 U/g glucose oxidase and 75 U/g catalase at 50 °C. The pH of the solution was maintained in the range of 6.8 ± 0.2. In the final crude ammonium gluconate-containing liquid, the protein, total nitrogen, total carbon, organic carbon, and ammonium salt content was 3.5 g/L, 8.47 g/L, 53.62 g/L, 13.79 g/ L, and 60 g/L, respectively. The environmental pollution was solved by using potato starch to produce value-added products.

The ubiquitin-proteasome system inkidney physiology and disease.

Intracellular proteins continuously turn over by degradation and synthesis in all organ tissues. Owing to its irreversible nature, protein degradation is a highly selective process to avoid irreparable breakdown of cellular constituents, thereby disrupting cellular stability, integrity and signalling. The majority of intracellular proteins are degraded by the ubiquitin-proteasome system (UPS), a multi-enzyme process that involves the covalent conjugation of ubiquitin to a substrate protein and its recognition and degradation by the core multicomponent proteolytic complex of the UPS, the proteasome. In addition to labelling misfolded, damaged, aggregation-prone and intact but unneeded proteins for proteasomal degradation, ubiquitylation regulates a multitude of cellular processes, such as transcription, translation, endocytosis, and receptor activity and subcellular localization. In addition, the proteasome generates peptides for antigen presentation in the immune system and for further degradation by peptidases to provide amino acids for protein biosynthesis and gluconeogenesis. Alterations of the UPS or of protein substrates that render them more or less susceptible to degradation are responsible for disorders associated with renal cell dysfunction. In this Review, we provide insight into the elegant and complex nature of UPS-mediated proteostasis and focus on its established and potential roles in renal cell physiology and pathophysiology.

13C-Labeled-Starch Breath Test in Congenital Sucrase-isomaltase Deficiency.

Human starch digestion is a multienzyme process involving 6 different enzymes: salivary and pancreatic α-amylase; sucrase and isomaltase (from sucrose-isomaltase [SI]), and maltase and glucoamylase (from maltase-glucoamylase [MGAM]). Together these enzymes cleave starch to smaller molecules ultimately resulting in the absorbable monosaccharide glucose. Approximately 80% of all mucosal maltase activity is accounted for by SI and the reminder by MGAM. Clinical studies suggest that starch may be poorly digested in those with congenital sucrase-isomaltase deficiency (CSID). Poor starch digestion occurs in individuals with CSID and can be documented using a noninvasive C-breath test (BT). C-Labled starch was used as a test BT substrate in children with CSID. Sucrase deficiency was previously documented in study subjects by both duodenal biopsy enzyme assays and C-sucrose BT. Breath CO2 was quantitated at intervals before and after serial C-substrate loads (glucose followed 75 minutes later by starch). Variations in metabolism were normalized against C-glucose BT (coefficient of glucose absorption). Control subjects consisted of healthy family members and a group of children with functional abdominal pain with biopsy-proven sucrase sufficiency. Children with CSID had a significant reduction of C-starch digestion mirroring that of their duodenal sucrase and maltase activity and C-sucrase BT. In children with CSID, starch digestion may be impaired. In children with CSID, starch digestion correlates well with measures of sucrase activity.

Experimental Evolution of Trichoderma citrinoviride for Faster Deconstruction of Cellulose.

Engineering faster cellulose deconstruction is difficult because it is a complex, cooperative, multi-enzyme process. Here we use experimental evolution to select for populations of Trichoderma citrinoviride that deconstruct up to five-fold more cellulose. Ten replicate populations of T. citrinoviride were selected for growth on filter paper by serial culture. After 125 periods of growth and transfer to fresh media, the filter paper deconstruction increased an average of 2.5 fold. Two populations were examined in more detail. The activity of the secreted cellulase mixtures increased more than two-fold relative to the ancestor and the largest increase was in the extracellular β-glucosidase activity. qPCR showed at least 16-fold more transcribed RNA for egl4 (endoglucanase IV gene), cbh1 (cellobiohydrolase I gene) and bgl1 (extracellular β-glucosidase I gene) in selected populations as compared to the ancestor, and earlier peak expressions of these genes. Deep sequencing shows that the regulatory strategies used to alter cellulase secretion differ in the two strains. The improvements in cellulose deconstruction come from earlier expression of all cellulases and increased relative amount of β-glucosidase, but with small increases in the total secreted protein and therefore little increase in metabolic cost.

Synthesis of enantiopure l-(5-phenylfuran-2-yl)alanines by a sequential multienzyme process

The enantioselective synthesis of unnatural amino acids is an attractive goal. Increasing attention has been given in recent years to the development of dynamic kinetic resolution processes, providing the desired enantiomer in almost quantitative yields and with high enantiopurity. Herein we describe an efficient sequential multi-enzyme process for the preparation of enantiopure 5-phenylfuran-2-ylalanines l-4a–d, starting from racemic 2-acetamido-3-(5-phenylfuran-2-yl)propanoic acids rac-1a–d. The first step, the CaL-B-mediated dynamic kinetic resolution of the racemic oxazolones provided the N- and C-protected l-amino acids l-2a–d (81–92% ee) with 100% theoretical yield. The protecting groups were removed in excellent yields by a second (mild non-stereoselective PLE mediated hydrolysis of the ester) and a third (Acylase I catalyzed stereoselective hydrolysis of the amide) enzymatic step, thus increasing the enantiomeric excess of the target compounds over 99%.

Maximizing the efficiency of multienzyme process by stoichiometry optimization.

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one-pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes.

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA–D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB–DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC–DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.

Competition between 24:5n-3 and ALA for Δ6 desaturase may limit the accumulation of DHA in HepG2 cell membranes

The use of Delta 6 desaturase (D6D) twice in the conversion of alpha-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; 22:6n-3) suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 microM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of >18 microM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes.

Structure-Based Engineering of E. coli Galactokinase as a First Step toward In Vivo Glycorandomization

In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.

Mutation of SENP1/SuPr-2 Reveals an Essential Role for Desumoylation in Mouse Development

The covalent modification of proteins by the small ubiquitin-like protein SUMO has been implicated in the regulation of numerous biological processes, including nucleocytoplasmic transport, genomic stability, and gene transcription. Sumoylation occurs by a multienzyme process similar to ubiquitination and, in Saccharomyces cerevisiae, is reversed by desumoylating enzymes encoded by the Ulp1 and Smt4/Ulp2 genes. The physiological importance of desumoylation has been revealed by mutations in either gene, which lead to nonoverlapping defects in cell cycle transition and meiosis. Several mammalian Ulp homologues have been identified, but, to date, nothing is known of the phenotypic effects of their loss of function. Here, we describe a random retroviral insertional mutation of one homolog, mouse SENP1/SuPr-2. The mutation causes increased steady-state levels of the sumoylated forms of a number of proteins and results in placental abnormalities incompatible with embryonic development. Our findings provide the first insight into the critical importance of regulating sumoylation in mammals.

Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism.

In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2(+) led to chromosome fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.

Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species.

The biosynthesis of aflatoxins (B(1), G(1), B(2), and G(2)) is a multi-enzyme process controlled genetically by over 20 genes. In this study, we report the identification and characterization of the avfA gene, which was found to be involved in the conversion of averufin (AVF) to versiconal hemiacetal acetate (VHA), in Aspergillus parasiticus and A. flavus; a copy of avfA gene was also cloned from a non-aflatoxin producing strain A. sojae. Complementation of an averufin-accumulating, non-aflatoxigenic mutant strain of A. parasiticus, SRRC 165, with the avfA gene cloned from A. flavus, restored the ability of the mutant to convert AVF to VHA and to produce aflatoxins B(1), G(1), B(2), and G(2). Sequence analysis revealed that a single amino acid replacement from aspartic acid to asparagine disabled the function of the enzyme in the mutant strain SRRC 165. The A. parasiticus avfA was identified to be a homolog of previously sequenced, but functionally unassigned transcript, stcO, in A. nidulans based on sequence homology at both nucleotide (57%) and amino acid (55%) levels. In addition to avfA, another aflatoxin pathway gene, omtB, encoding for an O-methyltransferase involved in the conversion of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST), was cloned from A. parasiticus, A. flavus, and A. sojae. The omtB gene was found to be highly homologous to stcP from A. nidulans, which has been reported earlier to be involved in a similar enzymatic step for the sterigmatocystin formation in that species. RT-PCR data demonstrated that both the avfA and avfA1 as well as omtB genes in A. parasiticus were expressed only in the aflatoxin-conducive medium. An analysis of the degrees of homology for the two reported genes between the Aspergillus species A. parasiticus, A. flavus, A. nidulans and A. sojae was conducted.

The in Vitro Conversion of Norsolorinic Acid to Aflatoxin B1. An Improved Method of Cell-Free Enzyme Preparation and Stabilization

The biosynthesis of the environmental carcinogen aflatoxin B1 (13) is initiated by the formation of a C6-primer by a dedicated yeast-like fatty acid synthase. Homologation of this starter unit by a polyketide synthase gives the anthraquinone norsolorinic acid (2). Approximately 15 chemical steps follow from this first stable intermediate to the mycotoxin (13) itself. A new protocol of cell-free enzyme preparation has been developed from the fungus Aspergillus parasiticus which carries out all of these transformations for the first time. The key experimental step involves rapid concentration and efficient dialysis by membrane filtration to remove primary and secondary metabolites, cofactors, and small biomolecules (MW < 10 000). All enzymes of the aflatoxin biosynthetic pathway have been dramatically stabilized by this procedure, and the effects of added substrates and cofactors can be assayed against virtually no background reactions. The overall pathway from norsolorinic acid (2) to aflatoxin B1 (13) has been investigated, cofactor requirements defined for each step, and a time-course run in which only versicolorin A (9) and sterigmatocystin (11) were observed to accumulate. The post-bisfuran skeletal rearrangement of versicolorin A (9) to demethylsterigmatocystin (10) was studied in O-methylsterigmatocystin (12) in the presence of D2O and/or d7-glucose or stereospecifically labeled NADPD. Unexpectedly high extents of proton exchange were found in the A ring during this transformation, including at a site of formal reduction. A tentative mechanism is discussed to account for this multi enzyme process.