2,3-Dihydro-2,5-dihydroxy-4H-benzopyran-4-one: A Nonphysiological Substrate for Fungal Melanin Biosynthetic Enzymes

Thompson, James E.; Basarab, Gregory S.; Pierce, James C.; Hodge, C. Nicholas; Jordan, Douglas B.

doi:10.1006/abio.1997.2489

Cited by 24 publications

(31 citation statements)

References 18 publications

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“…With the improved binding of the inhibitor by the mutants, we next addressed the catalytic activity of the NTF2 mutants. Activity was determined by monitoring the dehydration of a scytalone analogue, DDBO (17) (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…Ligand binding assays were performed by measuring quenching of intrinsic protein fluorescence at 340 nm upon excitation at 290 nm using an SLM-Aminco (Urbana, IL) 8000 spectrofluorometer. Continuous assays of enzymatic activity were performed essentially as described by using DDBO as substrate (17). Product formation was followed by determining the change of absorbance at 320 nm (⌬ ϭ 1.5 mM Ϫ1 ⅐cm…”

Section: Methodsmentioning

confidence: 99%

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Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

Nixon

Firestine

Salinas

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The generation of enzymes to catalyze specific reactions is one of the more challenging problems facing protein engineers. Structural similarities between the enzyme scytalone dehydratase with nuclear transport factor 2 (NTF2) suggested the potential for NTF2 to be re-engineered into a scytalone dehydratase-like enzyme. We introduced four key catalytic residues into NTF2 to create a scytalone dehydratase-like active site. A C-terminal helix found in scytalone dehydratase but absent in NTF2 also was added. Mutant NTF2 proteins were tested for catalytic activity by using a spectroscopic assay. One of the engineered enzymes exhibited catalytic activity with minimal k cat and K m values of 0.125 min ؊1 and 800 M, respectively. This level of catalytic activity represents minimally a 150-fold improvement in activity over the background rate for substrate dehydration and a dramatic step forward from the catalytically inert parent NTF2. This work represents one of the few examples of converting a protein scaffold into an enzyme, outside those arising from the induction of catalytic activity into antibodies.One of the most alluring promises of protein engineering is the ability to create a biocatalyst for any given chemical reaction. Attempts using a combination of site-directed mutagenesis guided by structural and biochemical information have been somewhat successful (1). Even so, the creation of novel biocatalysts remains a challenging problem (2).Given that the current understanding of how one might create a potent biocatalyst is limited, we have proposed a classification that ranks the enzyme engineering efforts in terms of three levels of increasing difficulty (1). The first category includes the modification of the substrate or cofactor specificity of a given enzyme while retaining the catalytic activity of the enzyme scaffold. Examples of successful applications of this approach are: the modification of the substrate specificity of subtilisin (3-5), the generation of chymotrypsinlike specificity within trypsin (6), and the change in the cofactor specificity of glutathione reductase (7) and lipoamide dehydrogenase (8). The second level envisions the introduction of residues that will confer catalytic activity on a binding protein. This level recently has been accomplished by the re-engineering of cyclophilin into a proline-specific endopeptidase (9). A catalytic triad similar to that found in serine proteases was grafted into the peptidyl-prolyl binding cleft. The resultant protein had an efficiency (k cat ͞K m ) of 0.7 ϫ 10 4 M Ϫ1 ⅐s Ϫ1toward X-Pro dipeptide substrates and a rate enhancement of (k cat ͞k uncat ) 8 ϫ 10 8 fold. The third, and most challenging group, imagines taking a protein scaffold, devoid of catalytic and substrate binding activities, and introducing residues that convert the scaffold into a catalyst.Previously, we focused our efforts on the creation of hybrid enzymes through the fusion of protein modules joining a substrate binding domain and a catalytic domain (10). Although this appro...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

Nixon

Firestine

Salinas

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…The enzyme acts independently of cofactors, prosthetic groups, or metal ions (37). In addition to its natural substrates, scytalone and vermelone, SD is also able to act on the pyrone 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), which resembles the structure of YWA1 (38). Although both SD and AurZ catalyze dehydration reactions of related polycyclic substrates, they have limited primary sequence identity (13% when compared with SD from M. grisea) but a high level of similarity in the predicted secondary structure, suggesting a similar tertiary structure (47% when compared with SD from M. grisea).…”

Section: Discussionmentioning

confidence: 99%

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Frandsen

Schütt

Lund

et al. 2011

Journal of Biological Chemistry

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Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated ␥-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to-aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.The plant pathogenic fungus Fusarium graminearum (teleomorph Gibberella zeae) is capable of producing a plethora of secondary metabolites, many of which belong to the polyketide class of compounds, such as the mycotoxins zearalenone, fusarin C, and aurofusarin (1). The F. graminearum genome encodes 15 polyketide synthases (PKS) 2 (2, 3), of which FgPKS12 is the progenitor of nor-rubrofusarin/rubrofusarin/aurofusarin (4, 5), FgPKS4 and FgPKS13 are the progenitors of zearalenone (1, 6), FgPKS10 is the progenitor of fusarin C (7, 8), and FgPKS3 is the progenitor of an uncharacterized black/purple perithecial pigment (8). PKS genes are typically found in gene clusters that comprise genes encoding transcription factor, transporters, and tailoring enzymes required for biosynthesis of the final product (1, 4 -6). In addition to genes encoding well characterized classes of enzymes, the clusters typically also include orphan genes that encode proteins annotated as "hypothetical" or "conserved hypothetical proteins," signifying that no similarity to any previously characterized protein has been found. In the case of F. graminearum, more than half of all gene models found in the Fusarium graminearum Genome Database from the Munich Information Center for Protein Sequences (MIPS) fall into either of these two categories (9).Functional characterization of hypothetical proteins is challenging, but those belonging to secondary metabolite gene clusters provide a unique opportunity because their location suggests a function in the respective biosynthetic pathways. The PKS12 gene cluster in F. graminearum, which is responsible for biosyn...

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“…Materials and General Methods-DDBO was synthesized as described (29). Scytalone was purified from cultures of Rsy Ϫ mutants of M. grisea (25).…”

Section: Methodsmentioning

confidence: 99%

“…Additionally, we report that the new reductase is considerably more specific for 4HN over 3HN in comparison to 3HNR. Historically, the enzyme we denote as 3HNR (2,5,6,24,29,30,34) has been referred to by others (26 -28) and in the enzyme nomenclature data base as tetrahydroxynaphthalene reductase, and this discrepancy is resolved.…”

mentioning

confidence: 99%

The Second Naphthol Reductase of Fungal Melanin Biosynthesis inMagnaporthe grisea

Thompson

Fahnestock

Farrall

et al. 2000

Journal of Biological Chemistry

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148

153

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Mutants of Magnaporthe grisea harboring a defective gene for 1,3,8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6,8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway. The second naphthol reductase gene was cloned from M. grisea by identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase. The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase. The second naphthol reductase was produced in Esherichia coli and purified to homogeneity. Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2. On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase. Tetrahydroxynaphthalene reductase has a 200-fold larger K i for the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide. M. grisea mutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.

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2,3-Dihydro-2,5-dihydroxy-4H-benzopyran-4-one: A Nonphysiological Substrate for Fungal Melanin Biosynthetic Enzymes

Cited by 24 publications

References 18 publications

Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

The Second Naphthol Reductase of Fungal Melanin Biosynthesis inMagnaporthe grisea

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