The Second Naphthol Reductase of Fungal Melanin Biosynthesis inMagnaporthe grisea

Mutants of Magnaporthe griseaharboring 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.

Trihydroxynaphthalene (3HN) 1 reductase is the biochemical target of three commercial agricultural fungicides (tricyclazole, pyroquilon, and phthalide of Fig. 1) that are applied to prevent blast disease in rice (1)(2)(3)(4)(5)(6)(7)(8). 3HNR, a member of the enzyme super family known as the short chain dehydrogenases (9,10), catalyzes the reduction of 1,3,8-trihydroxynaphthalene to vermelone, an intermediate step in fungal melanin biosynthesis ( Fig. 1). In Magnaporthe grisea, the causal agent of rice blast, fungal melanin biosynthesis proceeds through a pentaketide route, joining acetate units to make 1,3,6,8-tetrahydroxynaphthalene (4HN) (7,8). 4HN is transformed to 1,8-dihydroxynaphthalene through a succession of two reduction and two dehydration steps. 1,8-Dihydroxynaphthalene is considered to be the ultimate precursor of fungal melanin, a polymer that is employed by the pathogen during the initiation of disease (11,12). Both dehydration reactions in the biosynthetic pathway are catalyzed by scytalone dehydratase (SD (13)(14)(15)), an enzyme that is also a target of a commercial fungicide (16 -19), and others that are in development (20 -23). X-ray structures of 3HNR, a homotetramer of 120 kDa, in the absence and presence of the inhibitor tricyclazole have been reported (5,6,24), and they provide the initial basis for a structure-based design program for inhibitors of the enzyme function.
Genetic studies on fungal melanin biosynthesis in M. grisea yielded three classes of melanin-deficient mutants based on pigmentation phenotypes (25). Instead of the black polymer of fungal melanin, Buf Ϫ mutants lacking catalytic activity for 3HNR produce buff-colored pigment, and Rsy Ϫ mutants lacking catalytic activity for SD produce rosy-colored pigment. Accumulated pigments in the mutants result from the oxidation products of the respective precursors of the enzyme in the pathway, 3HN in the Buf Ϫ mutants, and 4HN in the Rsy Ϫ mutants. Since the Buf Ϫ mutant accumulates oxidation products of 3HN, it is obvious when considering the procession of the pathway (Fig. 1) that there should be another biochemical route to produce 3HN from 4HN without using 3HNR as the catalyst. Another indication of a second reductase is that the fungicides used for preventing rice blast (tricyclazole, pyroquilon, and phthalide) are known to cause the accumulation of 3HN byproducts in fungal cultures (1,3,4,7,8). A more quantitative assessment of the situation is obtained from measuring the accumulation in the media of scytalone, which is considerably more stable than 3HN or 4HN (Table I). The wild-type and the Buf Ϫ mutant do not accumulate scytalone in their growth media, whereas the Rsy Ϫ mutant does. Crosses between the Buf Ϫ and Rsy Ϫ mutants yield double mutants that accumulate scytalone in their growth medium to about half the extent as the Rsy Ϫ mutant. These results also suggest that there is either another reductase responsible for catalyzing the reduction of 4HN to scytalone or there is another route alto-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The gether for producing scytalone in M. grisea. In this work we report that, indeed, there is another reductase in the pathway and that its presence in M. grisea fully accounts for the accumulation of scytalone in the double mutant (buf Ϫ rsy Ϫ ) described above. 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.

EXPERIMENTAL PROCEDURES
Materials and General Methods-DDBO was synthesized as described (29). Scytalone was purified from cultures of Rsy Ϫ mutants of M. grisea (25). The enzymatic synthesis of vermelone from scytalone was accomplished essentially as described (26). UV-visible spectrophotometric analyses were performed on HP 8452A or HP 8453 diode array spectrophotometers (Hewlett Packard). SDS-polyacrylamide gel electrophoresis analyses of proteins were conducted by using a PhastSystem (Amersham Pharmacia Biotech). Homogeneous 3HNR was purified as described (30). 3HNR subunit concentrations were estimated using a calculated ⑀ 280 value of 20,760 M Ϫ1 cm Ϫ1 . The molecular mass of 4HNR was determined using a Fisons VG Quattro II mass spectrometer with calibration against horse heart myoglobin as an external standard. M. grisea and its mutants were grown in liquid media as described (25), and the scytalone concentrations in the media were determined by using scytalone dehydratase in an end point spectrophotometric determination (13).
Preparation of 4HNR-A ZipLox (Life Technologies, Inc.) library containing cDNA from M. grisea strain 6043 (31) was probed with radiolabeled 3HNR cDNA, and a class of weakly hybridizing clones was isolated in addition to the homologous 3HNR clones. 2 Sequencing of the weakly hybridizing cDNAs confirmed identification of a putative second reductase (HNR) gene. Genomic HNR clones were identified from a phage genomic excision library (31) containing DNA from strain 4091-5-8 in the FIXBAR vector. A full-length cDNA coding sequence (pCB1346) was assembled into vector pTrc99A (Amersham Pharmacia Biotech) using polymerase chain reaction according to standard methods (32).
For expression in Escherichia coli, the 4HNR-coding sequence of pCB1346 was transferred as an NcoI-BamHI fragment into the vector pET-11d (Novagen), forming plasmid pFA127, where 4HNR expression is driven by a lac-controlled phage T7 promoter system. Expression levels in E. coli BL21(DE3) were unexpectedly low as a consequence of mRNA secondary structure formed by complementarity between the initial coding sequence and the upstream ribosome binding sequence provided by the vector. To circumvent this limitation without altering the 4HNR amino acid sequence, we employed a "ribosome delivery" strategy modeled after several natural systems in which translation of adjacent cistrons is coupled (33). We provided as a ribosome feeder upstream from the 4HNR initiation codon a highly translated mini-cistron (17 codons) derived from the N-terminal fragment of T7 gene 10 ( Fig. 2). This vector (pFA156) was constructed by inserting a doublestranded synthetic oligonucleotide with the overlapping translational stop and restart signals into pET21c(ϩ) (Novagen) in place of the multiple cloning site sequences (between BamHI and XhoI sites). The 4HNR coding sequence was inserted into pFA156 as an NcoI-ScaI fragment from pFA127 to form the expression plasmid pFA168. The 4HNR coding sequence in pFA168 (and pCB1346) was found to carry a sequence alteration, presumably introduced during polymerase chain reaction cloning, resulting in a substitution of Leu (CTC) for the natural Phe (TTC), found in the genomic clone, at position 135. This mutation was corrected by site-directed mutagenesis using the QuikChange kit (Stratagene). The entire coding sequence was verified by DNA sequencing.
E. coli BL21(DE3) cells transformed with the corrected pFA168 were grown to mid-log phase, induced 4 h with 1 mM 1-(isopropylthio)-␤-Dgalactopyranoside, harvested, resuspended in 25 mM Hepes-NaOH, pH 7.5, and lysed by sonication. Protein purification steps were at 0 -4°C. After centrifugation, the cleared lysate was brought to 25% of saturation with respect to (NH 4 ) 2 SO 4 and centrifuged. The supernatant was subjected to hydrophobic interaction chromatography on a phenyl-Sepharose fast flow column (Amersham Pharmacia Biotech) equilibrated with 25 mM Hepes-NaOH, pH 7.5, containing 25% of saturation (NH 4 ) 2 SO 4 . The column was developed with a linear gradient of equilibration buffer to equilibration buffer lacking (NH 4 ) 2 SO 4 . Fractions containing 4HNR were dialyzed exhaustively against 25 mM Hepes-NaOH, pH 7.5. The protein was subjected to anion exchange chromatography (Mono-Q, Amersham Pharmacia Biotech) with a linear gradient (0 to 0.5 M NaCl in 50 mM Tris-HCl, pH 7.5) to obtain homogeneous 4HNR. 4HNR subunit concentrations were estimated using a calculated ⑀ 280 value of 23,830 M Ϫ1 cm Ϫ1 .
Fungal Strains, Mutant Isolation-Wild-type strain 4091-5-8, the Rsy Ϫ mutant CP485, and the Buf Ϫ mutant CP61 were described previously (25). Preparation of the remaining strains will be described in detail elsewhere. 2 Briefly, the buf Ϫ rsy Ϫ double mutants 4442Ϫ4-3 and 4442-4-6 represent both pairs of double recombinant progeny from an nonparental ditype tetrad isolated from a genetic cross between Buf Ϫ mutant CP2833 and a Rsy Ϫ strain derived from CP485. The Buf Ϫ mutants CP2831 and CP2833 were constructed by gene disruption techniques to ensure a total the absence of 3HNR enzyme activity for these studies. Specifically, the internal 3HNR gene sequence encoding amino acids 39 -253 was replaced by the BAR gene, which confers bialaphos resistance to these mutants. A gene replacement vector for the 4HNR gene was produced by inserting the HPH gene, conferring hygromycin resistance, into an internal NotI site in the 4HNR coding sequence, and this vector was used to disrupt the 4HNR locus by homologous recombination. This 4HNR disruption vector was transformed into strain 4091-5-8 to produce the hnr Ϫ strain CP3097 and into the buf Ϫ rsy Ϫ double mutant 4442-4-3 to produce triple mutants CP3100, CP3101, and CP3102. The buf Ϫ hnr Ϫ double mutant was obtained from tetrad analysis of a cross between the hnr Ϫ gene disruption strain CP3097 and buf Ϫ strain CP61.
Determination 25 mM ethanolamine, and 100 mM NaCl at pH 7.0 and 25°C. Initial rates were measured spectrophotometrically after initiating reactions with enzyme. The following net extinction coefficients combining the oxidation of the varied substrates with the reduction of NADP ϩ (all in units of mM Ϫ1 cm Ϫ1 ) were used: DDBO, ⌬⑀ 340 ϭ 5.4; scytalone, ⌬⑀ 355 ϭ 9.38; vermelone, ⌬⑀ 340 ϭ 14.1. Reactions (1 ml) with PQ contained 200 M NADPH in 100 mM MOPS-NaOH and 1% Me 2 SO at pH 7.0 and 25°C. Reactions were initiated with PQ. Initial rates were monitored spectrophotometrically using ⌬⑀ 340 ϭ -6.2 mM Ϫ1 cm Ϫ1 (reduced PQ autooxidizes back to PQ rapidly relative to the time of the assay and does not contribute to the change in absorbance). Initial rate data were fitted to Equation 1 using Kinetasyst (IntelleKinetics) or Grafit 4.0 (Erithacus Software); is the observed velocity, k cat is the maximum velocity per enzyme protomer, K m is the Michaelis constant, and S is the varied substrate concentration. Relative substrate specificities for scytalone and vermelone, scytalone vermelone , were calculated through Equation 2 using the parameters determined with Equation 1; (k cat /K m ) vermelone is the specificity constant of the enzyme for vermelone, and (k cat /K m ) scytalone is the specificity constant of the enzyme for scytalone. Inhibition constants were determined in PQ reactions (60 s) that contained 2.5, 5.0, or 20 M PQ, and rate data were fitted to Equation 3, where I and K i are the inhibitor concentration and the dissociation constant for the inhibitor from the ternary complex (enzyme-NADPH-inhibitor), respectively. Inhibitor concentrations exceeded enzyme concentrations in the incubations by at least 4-fold.
Determination of Relative Substrate Specificities (Substrate-partitioning Method)-A multiwavelength spectrophotometric method (34) was used to determine the partitioning of vermelone and scytalone to their respective oxidized (naphthol) products in incubations that contained both substrates. Reactions (1 ml) having different molar ratios of scytalone and vermelone contained 50 M NADP ϩ in 50 mM MES, 25 mM Tris, 25 mM ethanolamine, and 100 mM NaCl at pH 7.0 and 25°C. Initial rates were measured spectrophotometrically after initiating reactions with 3HNR or 4HNR. Net extinction coefficient changes for scytalone oxidation and NADP ϩ reduction (in units of mM Ϫ1 cm Ϫ1 ) were ⌬⑀ 244 ϭ 5.7; ⌬⑀ 282 ϭ Ϫ4. 8 . Equilibrium constants were determined in 100 mM sodium phosphate at pH 7.0 and 25°C. K eq (scytalone, 4HN) determinations were in 1-ml reactions containing 70 M scytalone and 29, 58, 116, or 290 M NADP ϩ . Reactions were initiated with 3HNR (80 g in 2 l) and monitored until the absorbance at 348 nm was constant. A net extinction coefficient for products minus reactants (⌬⑀ 348 ϭ 11.3 mM Ϫ1 cm Ϫ1 ) was used in calculating the equilibrium concentrations. K eq (vermelone, 3HN) determinations were in 1-ml reactions containing 106 M vermelone and 29, 58, or 116 M NADP ϩ . Reactions were initiated with 3HNR (80 g in 2 l) and monitored until the absorbance at 348 nm was constant. A net extinction coefficient for products minus reactants (⌬⑀ 348 ϭ 12.2 mM Ϫ1 cm Ϫ1 ) was used in calculating the equilibrium concentrations.

RESULTS
Cloning and Mutation Analysis of 4HNR in M. grisea-The putative second reductase gene was identified as an M. grisea gene with weak homology to the 3HNR gene. Mutants were produced by gene disruption techniques for phenotypic analysis. Hnr Ϫ mutants have similar pigmentation properties as the wild-type strains. However, hnr Ϫ buf Ϫ double mutants were found to develop a cherry red pigment in their growth medium, a pigmentation phenotype that resembles that of the Rsy Ϫ mutants and reflects the accumulation of oxidation products of 4HN. Whereas double mutants (buf Ϫ rsy Ϫ ) lacking SD and 3HNR functions accumulate significant quantities of scytalone in their growth media, triple mutants (buf Ϫ rsy Ϫ hnr Ϫ ) do not ( Table I). The triple mutants develop red pigmentation similar to the buf Ϫ hnr Ϫ double mutants. It is concluded that the second reductase gene encodes for a 4HNR capable of catalyzing the reduction of 4HN to scytalone and that the activity of this 4HNR fully accounts for the conversion of 4HN to scytalone in the buf Ϫ and buf Ϫ rsy Ϫ mutants.
Expression of M. grisea 4HNR in E. coli-The 4HNR gene product was produced in E. coli and purified to homogeneity. The predicted molecular mass of the product of the coding sequence is 28,637.5 Da. The molecular mass of the purified protein determined by electrospray ionization mass spectrometry is 28,436 Da. The protein was found to have the N-terminal sequence Pro-Ser-Ala-Asp lacking the N-terminal Met and Ala residues of the primary translation product, presumably as a consequence of the processing activity of methionine aminopeptidase and aminopeptidase P in E. coli (35). The predicted molecular mass of the aminopeptidase-processed protein is 28,435.2, in good agreement with the mass spectroscopy results. A sequence alignment of 4HNR with 3HNR proteins indicates that the two proteins are 46% identical (Fig. 3). Based on the x-ray structure of the 3HNR-NADPH-tricyclazole complex (5), there are nine amino acid side chains within a 4-Å sphere around the inhibitor, which occupies the naphthol bind-

FIG. 2. Ribosome delivery construct for expression of 4HNR.
An oligonucleotide (bold italics) inserted into pET21c(ϩ) provided a termination codon for the upstream truncated T7 gene 10 cistron and a ribosome binding site (q) for the 4HNR cistron embedded in the upstream coding sequence.  (Fig. 3). The catalytic triad of 3HNR (Ser-164, Tyr-178, and Lys-182) is conserved in 4HNR. Substrate Specificities of 3HNR and 4HNR-Steady-state kinetic parameters for substrates scytalone and vermelone were determined for 4HNR and 3HNR under the same conditions at pH 7.0 and 25°C (Table II). The individual determinations of substrate specificities for vermelone [k cat /K m (vermelone)] and scytalone [k cat /K m (scytalone)] suggest that the discrimination between the two enzymes is small and slightly favors the wrong selectivity according to our designations of the two enzymes based on the phenotypes of the M. grisea mutants. However, it is known that there are strong contributions from substrate inhibition in the determined values despite our efforts to diminish such effects, 3 and on this basis we must discount the specificity ratios. In the effort to determine true values for the relative substrate specificities, we employed a direct method for measuring substrate competition (34). In this procedure the dehydrogenation rates for vermelone and scytalone are determined simultaneously in a single incubation for the individual enzymes, 3HNR and 4HNR. From the values reported in Table II (Fig. 1), which more closely resembles vermelone than scytalone, is a much better substrate for 3HNR than 4HNR, whereas PQ (Fig. 1), which does not closely resemble either vermelone or scytalone, is accepted by 3HNR and 4HNR with similar substrate specificities.
Inhibitor Specificities of 3HNR and 4HNR-Inhibition constants were determined in the PQ reactions because NADPH potentiates the binding of tricyclazole (2) and the other inhibitors. K i values for three commercial fungicides (tricyclazole, pyroquilon, and phthalide) are listed in Table III. The inhibitors, particularly tricyclazole, have much greater affinity for 3HNR-NADPH than for 4HNR-NADPH.

Substrate Preferences of 3HNR and 4HNR-
The dilemma of an alternate route for the production of scytalone, as described in this study, originated from genetic studies and was addressed using a combination of molecular biology and biochemistry methods. Without doubt, 4HN is the precursor to scytalone. Also without doubt, there are two and only two naphthol reductases within the fungal melanin biosynthetic pathway. These conclusions are based on the results described in Table I where the triple mutants (lacking catalytic activities for 3HNR, 4HNR, and SD) do not accumulate scytalone. Even though their active-site residues are similar, the two reductases catalyze the same naphthol reduction reactions with considerably different substrate specificities.
We chose to examine the reverse reactions of the reductases using scytalone and vermelone because these substrates are significantly more stable than their oxidized (naphthol) counterparts. Neither 3HNR or 4HNR behave nicely in their steadystate reactions with vermelone or scytalone because of severe product-substrate inhibition. Rate limitations for the 3HNRcatalyzed oxidation of scytalone have been attributed to product release (2). We have found that 3HN forms a tight dead end complex with 3HNR-NADP ϩ (K i ϭ 60 nM), 3 and this accounts for part of the problem in determining rate constants. It is interesting to speculate that 3HN, which has a pK a of 5 and exists as an anion at pH 7, has an attraction for the positively charged pyridine ring of NADP ϩ as has been found with an anionic inhibitor of enoyl (acyl carrier protein) reductase complexed with NAD ϩ (36). As a result of the problems associated with determining steady-state kinetic parameters for scytalone and vermelone oxidation, the substrate specificities calculated from individually determined k cat /K m values suggest inaccurately that 3HNR prefers scytalone over vermelone and that 4HNR prefers scytalone over vermelone to a smaller extent than 3HNR (Table II). These results are not consistent with the genetics and highlight the difficulty of determining substrate specificities for 4HNR and 3HNR in this manner. 3 J. Thompson   Viviani et al. (26,27) and Vidal-Cros et al. (28) report cloning and characterization of a "tetrahydroxynaphthalene reductase" that varies from the 3HNR we have studied by three amino acids that lie outside the active site. We believe this difference owes to the two M. grisea strains used as the enzyme sources. Their gene was cloned by using the amino acid sequence of the protein purified from wild-type cultures of M. grisea (28). Our 3HNR gene was cloned by complementation of a Buf Ϫ mutant of M. grisea (30), so in vivo the gene product is the catalyst responsible for the conversion of 3HN to vermelone. The 99% sequence identity of their protein to ours makes it another 3HNR. Their description of the enzyme as a 4HNR was based on its substrate specificities for the naphthols, 3HN and 4HN. It is likely that their determination of kinetic parameters for the individual naphthol reductions also suffered from systematic errors owing to substrate and product inhibition.
In view of the problems involved in accurately determining k cat /K m values for the substrates of 3HNR and 4HNR, it was necessary to employ a direct competition method (34) to determine the true preferences of 3HNR and 4HNR for vermelone and scytalone. With this method, problems associated with nonproductive substrate binding and product inhibition are equalized because the two substrates are contained in the same incubation with the enzyme (37). The determinations from this method conclusively indicate that 3HNR prefers vermelone over scytalone by a factor of 39 and that 4HNR prefers scytalone over vermelone by a factor of 33 (Table II). Using Equation  6 with the equilibrium constants reported here shows that 3HNR prefers 3HN over 4HN by a factor of 4.2 and that 4HNR prefers 4HN over 3HN by a factor of 310. Thus, relative substrate specificities of the two enzymes for the two substrates differ by a factor of 1300.
Computer modeling of 3HN and 4HN into the active site of 3HNR suggests that there is steric and electrostatic repulsion between the sulfur atom of Met283 (C-terminal residue) and the C6 hydroxyl group of 4HN but not with 3HN, which has a hydrogen at C6, thus accounting for the preference of 3HNR for 3HN. 4 A homology model of 4HNR based on the x-ray structure of 3HNR does not show repulsion between the sulfur atom and the C6 hydroxyl group of 4HN because the C terminus of 4HNR is one residue shorter than that of 3HNR (Fig. 3); there is no methionine residue in the way, consistent with the better acceptance of 4HN by 4HNR than by 3HNR. The involvement of C-terminal residues in forming enzyme active sites has been documented in other systems (38 -40). Why 4HNR prefers 4HN over 3HN by a factor of 310 remains a mystery. High quality crystals of 4HNR have been obtained, 5 and perhaps a structural analysis of the protein will provide insights regarding its preference for 4HN. The enzyme nomenclature describes 3HNR as tetrahydroxynaphthalene reductase with a designation of EC 1.1.1.252. The results of this work require that our 3HNR be described as trihydroxynaphthalene reductase. Additionally, there should be a new entry for 4HNR as tetrahydroxynaphthalene reductase. 6 Inhibitors and the Biology of Fungal Melanin-The fungicides (tricyclazole, pyroquilon, and phthalide) target the function of 3HNR in the prevention of disease. When wild-type M. grisea is incubated with the inhibitors, the fungus accumulates 3HN and its oxidized byproducts. 4HNR has much lower affinity for the inhibitors than 3HNR (by factors of 200, 30, and 25  for tricyclazole, pyroquilon, and phthalide, respectively; Table  III), and this accounts for the accumulation of 3HN in the fungus instead of 4HN. At higher tricyclazole concentrations, the cherry red pigment characteristic of the hnr Ϫ buf Ϫ double mutant can appear in the wild-type, 7 consistent with inhibition of 4HNR activity at elevated levels of the fungicide.
Whereas the 3HNR gene from M. grisea is required for pathogenicity of the rice blast fungus (25), the 4HNR gene is not. 2 Certainly the activity of 4HNR is not absolutely required in cultures of M. grisea for the formation of fungal melanin as the hnr Ϫ mutant lacking the 4HNR gene has a wild-type phenotype. The catalytic activity of 3HNR must be responsible for reducing 4HN to scytalone in the mutants devoid of the 4HNR gene even though 3HNR has lower substrate specificity for 4HN in comparison with 3HN. In this work, we conclude that the catalytic activity of 4HNR is responsible for the production of scytalone in the double mutant buf Ϫ rsy Ϫ (lacking 3HNR and SD activities). Approximately half of the scytalone concentration is accumulated in the buf Ϫ rsy Ϫ double mutant in comparison to that of the rsy Ϫ single mutant (Table I), and this suggests that 4HNR has significant impact on flux through the melanin biosynthetic pathway.
Considered together, the genetic and biochemical data show that 4HNR might facilitate M. grisea infection, but that it is not required for it. Perhaps the activity of 4HNR in M. grisea is needed for the infection process under conditions that have not been examined. Otherwise, one might speculate that 4HNR and its gene are a conundrum of nature targeting geneticists and enzymologists. On the other hand, one might reflect that 4HNR is a potential element for providing resistance to commercial fungicides that target 3HNR. However, even though 4HNR has much lower affinities than 3HNR for the inhibitors (Table III), its relative specificity for 4HN over 3HN is 310, and its activity is not sufficient to support the reduction of 3HN to vermelone. If it were, the buf Ϫ mutant would have a wild-type phenotype. To date there are no reports of resistance to the 3HNR-targeted fungicides.
There are notable comparisons between the inhibitory complex formed by tricyclazole with 3HNR and the inhibitory complex formed by the antibacterial agent triclosan with its short chain dehydrogenase target enoyl (acyl carrier protein) reductase. The NAD ϩ form of the latter enzyme binds triclosan more strongly than the NADH or free enzyme forms (36). Ward et al. (36) point out that this is an advantage for the performance of the inhibitor in vivo because the NAD ϩ form of the enzyme is highly populated due to the 40-fold greater concentration of NAD ϩ over NADH in cells (36). 3HNR has the opposite disposition in that tricyclazole, and the other commercial inhibitors bind with the affinity progression 3HNR-NADPH Ͼ 3HNR-NADP ϩ Ͼ 3HNR (2). The inhibitors of 3HNR also select a highly populated form of the enzyme as the concentration of NADPH is higher in cells than that of NADP ϩ (41). On the basis of the equilibrium constants determined for this work, it is also clear that effective fungicides inhibit the thermodynamically more difficult of the two reduction reactions in the melanin biosynthetic pathway.