Manganese Peroxidase Isozyme Research Articles

Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata

The effects of high manganese [180 μM Mn(II)] concentration and addition of malonate (10 mM) were studied in nitrogen-limited cultures of the white-rot fungus, Phlebia radiata. High levels of manganese alone showed no systematic influence on the production of lignin peroxidase (LiP), manganese peroxidase (MnP) or laccase. In contrast, high-manganese containing cultures of P. radiata showed lower efficiency in the mineralization of 14C-ring-labelled synthetic lignin ([14C]DHP). The highest rates of mineralization, up to 30% in 18 days, were reached in low- manganese(2 μM)-containing cultures when malonate was omitted. Degradation of [14C]DHP was substantially restricted by the addition of malonate. The combination of high manganese and malonate resulted in increased levels of MnP and laccase production, whereas LiP production was repressed. Also, the profiles of expression of the MnP and LiP isozymes were affected. A new P. radiata MnP isozyme of pI 3.6 (MnP3) was found in the high-manganese cultures. Addition of malonate alone caused some repression but also stimulating effects on distinctive MnP and LiP isozymes. The results indicate that manganese and malonate are individual regulators of MnP and LiP expression and have different roles in the degradation of lignin by P. radiata.

The Effect of Veratryl Alcohol on Manganese Oxidation by Lignin Peroxidases

The extracellular peroxidase isozymes secreted by the white rot fungusPhanerochaete chrysosporiumhave been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported that lignin peroxidase isozyme H2 can also oxidize Mn2+(Khindariaet al.,1995,Biochemistry34, 7773–7779). This lignin peroxidase isozyme oxidized Mn2+with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H10) also readily oxidized Mn2+, but that the rate of Mn2+oxidation by compound II was extremely slow. Compound III rapidly formed in the presence of Mn2+, oxalate, and H2O2. However, upon the addition of veratryl alcohol, the results indicate that veratryl alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn2+oxidation was observed. The rate of Mn2+oxidation was the same as for the reduction of compound II by veratryl alcohol. The dependence of the rate of Mn2+oxidation on the concentration of veratryl alcohol was consistent with a mechanism in which Mn2+is oxidized by compound I and veratryl alcohol is oxidized by compound II. Therefore, under physiologically relevant conditions, in which both veratryl alcohol and Mn2+are present, all lignin peroxidase isozymes would be capable of oxidizing Mn2+to Mn3+which can serve as a diffusible oxidant.

Characterization of manganese(II) binding site mutants of manganese peroxidase.

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.

The Manganese Binding Site of Manganese Peroxidase: Characterization of an Asp179Asn Site-Directed Mutant Protein

A site-directed mutant, D179N, in the gene encoding Phanerochaete chrysosporium manganese peroxidase isozyme 1 (mnp1), was created by overlap extension, using polymerase chain reaction. The mutant gene was expressed in P. chrysosporium under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidase (MnP) was purified, and its spectra and MW were very similar to those of the wild-type enzyme. Steady-state kinetic analysis of MnP D179N revealed that the Km for the substrate MnII was approximately 50-fold greater than the corresponding Km for the wild-type recombinant enzyme (3.7 mM versus approximately 70 microM). Likewise, the kcat value for MnII oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent Km for H2O2 of MnP D179N was similar to the corresponding value of the wild-type MnP. The first-order rate constant for MnP D179N compound II reduction by MnII was approximately 1/200 of that for the wild-type enzyme. The equilibrium dissociation constant (KD) for MnP D179N compound II reduction by MnII was approximately 100-fold greater than the KD for the wild-type compound II. In contrast, the second-order rate constant for p-cresol reduction of the mutant compound II was similar to that of the wild-type enzyme. These results also suggest that the mutation affects the binding of MnII to the enzyme and, consequently, the rate of compound II reduction by MnII. In contrast, the mutation apparently does not have a significant effect on H2O2 cleavage during compound I formation or on p-cresol reduction of compound II.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a cDNA encoding a manganese peroxidase from Phanerochaete chrysosporium: genomic organization of lignin and manganese peroxidase-encoding genes

Two heme proteins, manganese peroxidase (MnP) and lignin peroxidase (LiP), play key roles in the fungal depolymerization of lignin. Many cDNA and genomic clones encoding these peroxidases have been published. We report here on the cDNA λMP-2 encoding the MnP isozyme H3 from Phanerochaete chrysosporium strain BKM-F-1767. We also demonstrate that the MnP-encoding gene, λMP-1, encoding isozyme H4, and λMP-2 reside on separate chromosomes from each other and from the LiP-encoding genes. From these results, it is apparent that λMP-2 is not linked to λMP-1 or other genes believed to be involved in lignin depolymerization, such as the LiP and glyoxal oxidase.

Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomycete Phanerochaete chrysosporium

The nucleotide (nt) sequence of a gene ( mnp2) encoding manganese peroxidase isozyme 2 (MnP-2) from Phanerochaete chrysosporium was determined. The sequence of 3297 bp includes 1287 bp of 5'-flanking sequence and 490 bp 3' to the stop codon. Comparison of cDNA and genomic sequences indicates seven introns varying in size from 50–55 bp. The 5' upstream region of the mnp2 gene contains a TATAA element, three inverted CCAAT elements (ATTGG), six putative heat-shock elements ( HSE) and three putative metal response elements ( MRE) (TGCRCNC), all located within 1100-bp upstream from the start codon. The positions of the putative HSE and MRE in the promoter region of mnp2 are compared with the corresponding sequences in the mnp1 gene promoter. A Northern blot probed with a fragment specific for the mnp2 gene suggests that the transcription of mnp2 is regulated by Mn ions.

Manganese peroxidases of the white rot fungus Phanerochaete sordida

The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene.

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.

Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium

The complete nucleotide (nt) sequence of a gene ( mnp-1) encoding manganese peroxidase isozyme 1 (MnP-1)(pI= 4.9) from Phanerochaete chrysosporium has been determined. The sequence of 2539 bp includes 526 bp of 5′-flanking sequence and 368 bp 3′ to the poly(A) site. Comparison of cDNA and genomic sequences indicates six introns varying in size from 57–72 bp. Intron splice-junction sequences all adhere to the GT---AG rule. The positions of the introns show little similarity to the intron positions in the closely related lignin peroxidase-encoding genes. The 5′ upstream region of the mnp-1 gene contains a TATAA elements and three inverted CCAAT elements (ATTGG) at nt positions −81, −181, −195, and −304, respectively, relative to the start codon. In addition, the mnp-1 gene contains three putative heat-shock (HS) elements similar to the consensus C--GAA--TTC--G sequence, and two consensus metal response elements located within 500 bp upstream from the start codon. Furthermore, Northern-blot analysis demonstrates that mnp gene transcription is regulated by HS.