Enzymes in Biomass Conversion - American Chemical Society

Chapter 15

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Structure and Regulation of Manganese Peroxidase Gene from Phanerochaete chrysosporium M . H . Gold, J . A. Brown, B. J . Godfrey, M . B. Mayfield, H. Wariishi, and Κ. Valli Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-1999 Manganese peroxidase (MnP) is one of two lignin -degrading enzymes secreted by the white-rot basidiomy cete Phanerochaete chrysosporium. MnP is an H O dependent heme peroxidase. The enzyme oxidizes Mn to Mn , which in turn oxidizes the terminal phenolic substrates. Several cDNAs encoding MnP isozymes have been isolated and characterized. A cDNA probe was used to isolate and characterize a gene encoding MnP. Comparison of the cDNA and genomic sequences reveal 6 introns, all containing less than 72 base pairs. The appearance of MnP activity in the extracellular medium depends on the presence of Mn. In addition, MnP mRNA is only detected in cells grown in the presence of Mn. If Mn is added to 4-day-old nitrogen-limited Mn -deficient cultures, extracellular MnP activity appears within 6 hours and MnP mRNA appears within 1 hour. These results indicate that Mn is involved in the transcriptional regulation of the MnP gene. 2

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Lignin, a heterogeneous, phenylpropanoid polymer, is the most abundant renewable aromatic material. It comprises 15-30% of woody plant cell walls, forming an encrusting matrix surrounding the cellulose, which retards the microbial depolymerization of that most prevalent natural polymer (1,2). Thus the degradation of lignin plays a key role in the earth's carBon cycle (1-3). White-rot basidiomycetous fungi are the only known organisms whicfi~are capable of degrading lignin completely to C 0 and H Ο in pure culture. The white-rot basidiomycete Phanerochaete chrysosporium degrades lignin during secondary metabolic (idiophasic) growth, the onset of which is triggered by limiting cultures for nutrient nitrogen (4-6). Under ligninolytic conditions P. chrysosporium secretes two extracellular heme peroxidases, manganese peroxidase (MnP) and lignin peroxidase (LiP) which, along with an H 0 generating system, are apparently the major components of its hgnm-degradative system (4-7). A 2

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0097-6156/91/0460-0188$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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considerable amount of evidence from model substructure studies suggests that Li Ρ and MnP are responsible for the initial degradation of lignin by this organism (4,6-8). The structure and mechanism of lignin peroxidase have been studied extensively (4,6,7,9,10). Structural and Catalytic Properties of Manganese Peroxidase The second ligninolytic enzyme, MnP, has been identified (Π), purified and characterized (13-16). MnP is an Ο -dependent heme glycoprotein (M -46,000) with an iron protoporphyrin ΓΧ prosthetic group. MnP catalyzes the Mn -dependent oxidation of a variety of phenols and phenolic lignin model compounds (6,8.13-15,17). Electronic absorption (6,13,14). EPR and resonance Raman (18) evidence indicates that the heme iron m the native protein is in the higrnspin, pentacoordinate, ferric state, suggesting that the heme environment is similar to that of horse radish peroxidase (HRP) (19). In recent work Q8), EPR spectra of i N O - and NO- adducts of ferrous MnP were compared with those of HRP. Coordination of a histidine as the fifth ligand to the heme iron (proximal His) was confirmed from the pattern of superhyperfme splittings of the NO signals centered at g « 2.005. It has been demonstrated that M n is the preferred substrate for MnP (13-17). The enzyme oxidizes M n to M n and the M n produced, complexed with a suitable carboxylic acid ligand (12-16), diffuses from the enzyme and in turn oxidizes the organic substrates (6,8,13-17). Thus the Mn ion participates in the reaction as a diffusible redox couple (Fig. 1) rather than as an enzyme-binding activator. In support of this concept, we have demonstrated that chemically prepared M n complexed with a carboxylic acid ligand such as malonate or lactate mimics the reactivity of the enzyme (6,8,14,15). Our spectral and kinetic characterization of the oxidized intermedi ates of MnP compound I (MnPI), and MnP compound II (MnPII) indicate (Fig. 1) that the oxidation states and catalytic cycle of MnP are similar to LiP and HRP (6,15,16,19). MnPI contains two oxidizing equivalents over the native feme enzyme. The first equivalent resides in the F e = 0 structure (19); the second equivalent resides in a porphyrin π-cation radical [PVJ7 Reduction of compound I by one equivalent yields MnPII (Fig. 1); spectral evidence indicates that MnPII contains an F e = 0 structure (15,16). Our kinetic evidence also suggests that carboxylic acid chelators such as lactate or malonate facilitate the dissociation of the M n from the enzyme (16). r

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Structure of the Manganese Peroxidase Gene A P. chrysosporium cDNA library was prepared in the expression vector XGTll using RNA isolated from ligninolytic mycelia (20). The first MnP cDNA encoding MnP isozyme 1 (MnP-1) (pi = 4.9) was isolated using polyclonal antibody prepared against purified MnP-1 and sequenced using the dideoxy chain termination method. The deduced mature protein contains 357 amino acids (aa) with a calculated M of 37,439. This is -81% of the experimentally determined molecular weigrït with the discrepancy due to glycosylation (6,17,20). The deduced mature protein is preceded by a 21-aa signal peptide. Southern blot analysis (20) indicates that MnP-1 is a member of a family of P. chrysosporium MnP genes, and subsequently a second MnP cDNA encoding a related gene was sequenced (21; Gold, M . H.; Mayfield, M . B.; Nipper, V. J.; Pribnow, D.. unpublished results). r



FeUI 111

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Figure 1. Catalytic cycle of manganese peroxidase. The M n pro duced is chelated by organic acids and the complex diffuses from the enzyme surface. This Mn -organic acid complex in turn oxidizes phenolic substructures in lignin and other substrates. AH = phenolic lignin substructure.

Compound I FeIV=0[Pi]

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Most plant peroxidases contain 2 His and an Arg which are essential for activity (22,23). Our recent EPR and resonance Raman evidence (18; see above) demonstrates the presence of a proximal His ligated to the nëme iron of MnP-1. Comparative sequence analysis (Fig. 2) indicates that the sequences flanking the proximal His and the distal His and Arg are conserved in the MnP-1 protein. In addition, very little variation is observed in the locations of the proximal His (residue 176) and the distal His and Arg (residues 46 and 42, respectively), suggesting that all of these peroxidases evolved from a common precursor. The MnP-1 cDNA clone was used to probe a P. chrysosporium genomic DNA library in the vector XEMBL-3, and a MnP-1 genomic clone was isolated and characterized (Godfrey, B. J.; Mayfield, M . B., Brown, J. A; Gold, M . H., Gene, in press). The sequence includes 2539 base pairs (bp). Comparison of the cDNA and genomic sequences reveal 6 introns (Fig. 3) varying in size from 57 to 72 bp. The intron splice junction sequences of the MnP-1 gene all adhere to the GT—AG rule. Despite the strong homology in the coding regions between the MnP-1 gene (20) and several LiP genes (24-26). there is little similarity in the locations of introns. Codons for the distal His (His 46) and distal Arg (Arg 42)~amino acid residues thought to be involved in the heterolytic cleavage of H 0 during MnP compound 1 formation ( 16,20-22,24,25)--are together in exon 3 in the MnP-1 gene, but are split by intron 2 in the LiP genes (Fie. 3). The codon for the proximal His (His 173) is located in exon 5 of the MnP-1 gene and in exon 6 of the LiP gene. MnP lacks the putative 7-aa propeptide ending in the dibasic aa pair Lys-Arg, found between the signal peptide and the N-terminus of the mature LiP proteins (27); the sequences encoding the MnP-1 signal peptide and N-terminus of the mature protein are on the same exon. In the LiP gene, the sequence encoding the signal peptide is separated from that encoding the propeptide by an intron (Fig. 3) (27). The 5' upstream region of the MnP-1 gene contains a TATAAA element 81 bp upstream of the translation initiation codon. In addition, three inverted CCAAT elements (ATTGG) (28) are found at positions -181, -195, and -304 with respect to the initiation codon. We are analyzing the 5' upstream sequence of the MnP-1 gene for other possible regulatory sequence elements. 2

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Regulation of the Expression of Manganese Peroxidase by Manganese The ligninolytic system of P. chrysosporium is expressed during secondary metabolic (idiophasic) growth, the onset of which is triggered by limiting cultures for nutrient nitrogen (4,5). LiP and MnP activity appear in the extracellular medium only during the secondary metabolic phase of growth (4,13,20,24). Northern blot analysis has demonstrated that the expression of LiP (24) and MnP (20,21) isozymes is controlled at the level of gene transcription by nutrient nitrogen. As discussed above, Mn is the primary substrate for MnP (13-15). Our results demonstrate that the expression of MnP is regulated by Mn and by nitrogen. As shown in Figure 4. accumulation of MnP activity in the extracellular medium of nitrogen-limited cultures depends on the presence of Mn, confirming previous results from our lab and others (29-32). In contrast, comparisons of the mycelial biomass production from cultures grown in the presence and absence of Mn indicate that Mn has no significant effect on primary growth (32). Addition of Mn to the cellfree extracellular medium of cultures grown in the absence of Mn does not



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Figure 2. Comparison of P. chrysosporium MnP-1 and other peroxi dases at regions near the proximal arid distal histidines. The peroxidase sequences used were manganese peroxidase (MnP) (20), cytochrome c peroxidase (CCP) (38). horseradish peroxidase (HRP) (39), and LiP (24). Identical ammo acids are enclosed in solid boxes, and similar amino acids are enclosed in dashed boxes. (Reproduced with permission from Ref. 20. Copyright 1989. American Society for Biochemistry and Molecular Biology, Inc.)

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Proximal histidine MnP 163 Ρ F jÊ~ V; V s IL" *Π A 166 LiP 160 E L |E L] V w !M ±j S HRP 164 S S !D Lj V A JL (S G D R |_E v! V A Ik CCP G

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Figure 3. Comparison of the structures of the MnP-1 and L1P-H8 genes (26,27). The structures of the genes from the translation initiation codons to the termination codons are shown. Exons are indicated by shading. The putative signal peptides and propeptide are indicated by crosshatching. The positions of the distal His (His 46 and 47), distal Arg (Arg 42 and 43), and proximal His (His 173 and 176) for MnP-1 and L1P-H8, respectively, are also indicated. (Reproduced with permission from Godfrey, B. J.; Mayfield, M. B.; Brown, J. Α.; Gold, M. H. Gene 1990, in press. Copyright 1990, Elsevier Science Publishers Β. V.)

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Figure 4. Effect of Mn supplementation on the appearance of extra cellular MnP activity. Nitrogen-limited cultures were grown from a conidial inoculation. Extracellular MnP activity from cultures grown in the presence (open triangles) or absence (open circles) of 180 μΜ MnS0 was assayed as described (13.32). Dry weights of mycelia grown in the presence (solid triangles) and absence (solid circles) of Mn were also determined. (Reproduced with permission from Ref. 32. Copyright 1990. American Society for Microbiology.) 4


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restore MnP activity, indicating that Mn is not acting as an activator of the enzyme. This was tested directly using Western blotting. MnP antiserum detected both extracellular and intracellular MnP only in cultures grown in the presence of Mn. This indicates that no active or inactive extracellular MnP protein is present in the absence of Mn, and that Mn is not regulating secretion of the enzyme. When Mn is added to cultures previously grown for 4 days in the absence of Mn, extracellular MnP accumulation ensues within 6 hours and reaches a maximum within 18 hours (Fig. 5). This rapid response to Mn suggests that the Mn effect is probably not indirect~for example, by control ling the rate of nitrogen depletion, and consequently the onset of secondary metabolism and production of MnP. The lack of effect of Mn on mycelial growth also indicates that Mn is not regulating the utilization of nitrogen. The rapid response to the addition of Mn to 4-day-old cultures suggests that the metal affects the synthesis of the MnP protein directly. The inhibitory effects of actinomycin D and cycloheximide (Fig. 5) suggest that Mn is specifically involved in the transcriptional control of the MnP-1 gene. The translational inhibitor cycloheximide would inhibit MnP synthesis regardless of whether Mn affects transcription or translation. However, the inhibitory effect of the RNA synthesis inhibitor actinomycin D on MnP induction (Fig. 5) strongly suggests that Mn is involved in the transcriptional control of MnP synthesis rather than affecting mRNA stability or translation. Northern blot analysis confirms that Mn exerts its effect at the level of transcription. MnP mRNA is detectable in 4-day-old cultures and reaches a maximum in 5-day-old nitrogen-limited cultures grown in the presence of 180 μΜ Mn (Fig. 6a). This is in agreement with the results obtained for MnP activity. In contrast, no MnP mRNA is detectable in cultures grown in the absence of Mn. In addition, when Mn is added to a concentration of 180 μΜ to 4-day-old cultures grown in the absence of Mn, Northern blot analysis shows that MnP mRNA appears within 1 hour (Fig. 6b). These results strongly indicate that Mn, the substrate of the enzyme, is regulating the transcription of the MnP gene. This rapid response to Mn supports our argument that Mn is acting directly to control gene transcription rather than indirectly by regulating the utilization of nitrogen. Clarification of the details of Mn regulation of MnP gene transcription will require further study. However, a model whereby M n " binds to and activates a specific transcription factor is an attractive possibility because of the high degree of specificity involved (32). Metal-ion-specific transcrip tion factors occur in the activation of the yeast metallothionein gene by Cu (33), in the activation of the Mer operon by Hg (34). and in the activation oFthe Fe regulation system in Escherichia coli (35). However, other less specific mechanisms for Mn induction are also possible. For example, Mn might regulate a secondary messenger such as cAMP. Mn-specific adenyl cyclases such as have been found in yeast (36) could be regulating cAMP levels. However, the latter mechanisms seem less likely because they would be expected to result in global responses affecting a variety of metabolic functions. We plan to use the MnP gene and the DNA transformation system which we have recently developed for P. chrysosporium (37) to elucidate the regulatory sequences and protein Tactors involved in the Mn control of MnP gene transcription.


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Figure 5. Effect of actinomycin D and cycloheximide on the induction of MnP activity. Mn-deficient cultures were grown for 4 days after which MnSO (180 μΜ) was added alone (triangles), simultaneously with actinomycin D (50 /ig/mL) (solid circles), or with cycloheximide (50 Mg/mL) (open circles). Extracellular enzyme activity was assayed at the indicated intervals after the additions, by the ABTS method (13,32). (Reproduced with permission from Ref. 32. Copyright 1990, American Society for Microbiology.)


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Figure 6. a. Northern blot analysis of P. chrysosporium RNA probed with P-labeled MnP-1 cDNA. RNA from Mn-dericient (Mn") and complete cultures (Mn ) was isolated, separated by electrophoresis, and probed as described (32). Samples were as follows, day 4, Mn" (lane 1); day 4, Mn (lane 2); day 5, Mn" (lane 3); day 5, Mn (lane 4); day 6, Mn" (lane 5); day 6, Mn (lane 6). (Reproduced with permission from Ref. 32. Copyright 1990, American Society for Microbiology.) b. Mn induction of MnP gene transcription. Mn-deficient cultures were grown for 4 days, after which MnS0 was added to the experimental cultures to a final concentration of 180 μΜ. One hour after the addition of Mn. total RNA was extracted, electrophoresed, transferred to filters and probed with P-labeled cDNA (32). These results indicate that Mn induces the transcription of the MnP-1 gene. 32

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Acknowledgments: Supported by grants FG06-87ER-13715 from the U. S. Dept. of Energy, Office of Basic Energy Sciences, and DMB 8904358 from the National Science Foundation. Literature Cited 1. Crawford, R. L. Lignin Biodegradation and Transformation; Wiley: New York, 1981. 2. Sarkanen, Κ. V.; Ludwig, C. H. Lignins. Occurrence, Formation, Structure and Reactions. Wiley-Interscience: New York, 1971. 3. Freudenberg, K. In Constitution and Biosynthesis of Lignin; Neish, A. C.; Freudenberg, K., Eds.; Springer-Verlag: New York, 1968, pp 47-122. 4. Kirk, T. K.; Farrell, R. L. Annu. Rev. Microbiol. 1987, 41, 465505. 5. Buswell, J. Α.; Odier, E. CRC Crit. Rev. Biotechnol. 1987, 6, 160. 6. Gold, M . H.; Wariishi, H.; Valli, K. In Biocatalysis in Agricultural Biotechnology; Whitaker, J. R.; Sonnet, P. E., Eds.; American Chemical Society: Washington, DC, 1989, ACS Symposium Series No. 389, pp 127-140. 7. Tien, M . CRC Crit. Rev. Microbiol. 1987, 15, 141-168. 8. Wariishi, H.; Valli, K.; Gold, M . H. Biochemistry; 1989, 28, 6017-6023. 9. Marquez, L.; Wariishi, H.; Dunford. H. B.; Gold, M . H. J. Biol. Chem. 1988, 263, 10549-10552 10. Wariishi, H.; Gold, M . H. J. Biol. Chem. 1990, 265, 2070-2077. 11. Kuwahara, M ; Glenn, J. K.; Morgan. Μ. Α.; Gold. M . H. FEBS Lett. 1984, 169, 247-250. 12. Wariishi, H. Valli, K.; Renganathan. V.; Gold. M . H. J. Biol. Chem. 1989, 264, 14185-14191. 13. Glenn, J. K.; Gold, M . H. Arch. Biochem. Biophys. 1985, 242, 329341. 14. Glenn, J. K.; Akileswaran. L.; Gold. M . H. Arch. Biochem. Biophys. 1986, 251, 688-696. 15. Wariishi, H.; Akileswaran, L.; Gold. M . H. Biochemistry 1988, 27, 5365-5370. 16. Wariishi, H.; Dunford, H. B.; MacDonald, I. D.; Gold. M. H. J. Biol. Chem. 1989, 264, 3335-3340. 17. Paszczynski, Α.; Huynh, V.-B.; Crawford, R. L. Arch. Biochem. Biophys. 1986, 244, 750-65. 18. Mino, Y . ; Wariishi, H.; Blackburn. N . J.; Loehr, T. M . ; Gold, M . H. J. Biol. Chem. 1988, 263, 7029-7036. 19. Dunford, H. B.; Stillman, J. S. Coord. Chem. Rev. 1976, 19, 187251. 20. Pribnow, D. G.; Mayfield, M . B.; Nipper, V. J.; Gold, M . H. J. Biol. Chem. 1989, 264, 5036-5040. 21. Pease, Ε. Α.; Andrawis, Α.; Tien. M . J. Biol. Chem. 1989, 264, 13531-13535. 22. Poulos, T. L.; Kraut, J. J. Biol. Chem. 1988, 255, 8199-8205. 23. Mazza, G.; Welinder, K. G. Eur. J. Biochem. 1980, 108, 481-489. 24. Tien. M . ; Tu, C.-P. D. Nature 1987, 326, 520-523. 25. de Boer, Η. Α.; Zhang. Y. Z.; Collins, C.; Reddy, C. A. Gene (Amst.) 1987, 60, 93-102.


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26. Smith, T. L.; Schalch. H.; Gaskell. J.; Covert. S.: Cullen, D. Nucleic Acids Res. 1988, 16, 1219. 27. Schalch, H.; Gaskell. J.; Smith. T. L.; Cullen. D. Mol. Cell. Biol. 1989, 9, 2743-2747. 28. Dynan, W. S.; Tjian, R. Nature 1985, 316, 774-778. 29. Glenn, J. K., Ph.D. Dissertation, 1986, Oregon Graduate Institute, pp. 132-142. 30. Bonnarme, P.; Jeffries, T. W. Appl. Environ. Microbiol. 1990, 56, 210-217. 31. Gold, M . H.; Brown, J. Α.; Glenn, J. K. J. Cell. Biochem. 1990, 14CS, 206. 32. Brown, J. Α.; Glenn, J. K.; Gold, M . H. J. Bacteriol. 1990, 172, 3125-3130. 33. Furst, P.; Hu, S.; Hackett, R.; Hamer, D. Cell 1988, 55, 705-717. 34. O'Halloran, T. V. in Metal Ions in Biological Systems; Sigel, H.; Sigel, Α., Eds.; Marcel Dekker, New York, 1989, pp. 105-146. 35. de Lorenzo, V.; Wee, S.; Herrero, M . ; Neilands, J. B. J. Bacteriol. 1987, 169, 2624-2630. 36. Londesborough, J. C.; Nurminen. T. Acta Chem. Scand. 1972, 26, 3396-3398. 37. Alic, M . ; Kornegay, J.; Pribnow, D. G.; Gold, M . H. Appl. Environ. Microbiol. 1989, 55, 406-411. 38. Kaput, J.; Goltz, S.; Blobel, G. J. Biol. Chem. 1982, 257, 1505415058. 39. Welinder, K. G. FEBS Lett. 1976, 72, 19-23. RECEIVED September 26,

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Enzymes in Biomass Conversion - American Chemical Society

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