PubMed Health⌕ Search

Biomedical subjects

M H Gold

Publications and source records attributed to M H Gold.

At least 19 recordsLinked to original sources
PubMed1997

Haloperoxidase activity of manganese peroxidase from Phanerochaete chrysosporium.

Manganese peroxidase (MnP) from Phanerochaete chrysosporium exhibits haloperoxidase activity at low pH. In the presence of hydrogen peroxide, MnP oxidizes bromide and iodide as measured by the formation of tribromide and triiodide complexes and the halogenation of various organic substrates. The optimum pHs for bromide and iodide oxidation are 2.5 and 3.0, respectively. Transient-state kinetic studies show that the reaction between MnP compound I and bromide or iodide occurs via a single two-electron step process, obeying second-order kinetics. The second-order rate constants for MnP compound I reduction by bromide and iodide are (4.1 +/- 0.2) x 10(3) and (1.1 +/- 0.1) x 10(5) m-1 s-1, respectively, at pH 3.0. MnP brominates a variety of aromatic substrates, including veratryl (3,4-di-methoxybenzyl) alcohol (I) to produce of 2-bromo-4,5-dimethoxybenzyl alcohol (II). MnP also hydrobrominates cinnamic acid (VI) to produce 2-bromo-3-hydroxyphenylpropionic acid (VII). With 3,4-dimethoxycinnamic acid (III) as the substrate, two bromination products are identified: trans-2-bromo-1-(3, 4-dimethoxyphenyl) ethylene (IV) and 2-bromo-3-(3, 4-dimethoxyphenyl)-3-hydroxypropionic acid (V). MnP also brominates 1,3-dicarbonyl compounds such as monochlorodimedone and malonic acid. Incubation of MnP with bromide and H2O2 in the absence of organic substrates results in enzyme inactivation. MnP binds halides to produce characteristic optical difference spectra. From these spectra, apparent dissociation constants at pH 3.0 are determined to be 0.13, 20, and 45 mm for fluoride, chloride, and bromide, respectively.

Basidiomycota↗
PubMed1997

Crystal structures of substrate binding site mutants of manganese peroxidase.

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of MnII to MnIII. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed MnII binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of MnII bound to the protein. Crystals grown in the absence of MnII, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess MnII exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major MnII binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the MnII binding site. The structure of the MnII binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.

Basidiomycota↗
PubMed1997

Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and coordination.

A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with 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 manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.

Basidiomycota↗
PubMed1997

Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium.

The gene encoding manganese peroxidase isozyme 3 (MnP3) from the white-rot basidiomycete Phanerochaete chrysosporium was cloned and sequenced. The mnp3 gene encodes a mature protein of 357 amino acids with a 25 amino-acid signal peptide. The amino acids involved in peroxidase function, as well as those forming the MnII binding site and those involved in disulfide bond formation, are conserved in the MnP3 sequence. The mnp3 gene has six introns, indicating that the sequenced P. chrysosporium mnp genes can be divided into three subfamilies on the basis of intron-exon structure. The mnp3 gene promoter contains putative metal response elements and heat shock elements which may be involved in the regulation of mnp gene transcription by Mn, the substrate for the enzyme, and by heat shock.

Amino Acid Sequence↗
PubMed1997

Truncated-gene reporter system for studying the regulation of manganese peroxidase expression.

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of Phanerochaete chrysosporium is regulated by Mn, heat shock (HS), and H2O2 at the level of gene transcription. We have constructed a homologous gene reporter system to further examine the regulation of two mnp genes, mnp1 and mnp2, encoding individual MnP isozymes. Internal deletions of 234 and 359 bp were made within the coding regions of the mnp1 and mnp2 genes, respectively. The truncated mnp genes were subcloned into the shuttle vector pOGI18, which includes the Schizophylum commune ade5 gene as a selectable marker, and transformed into a P. chrysosporium Ade1 auxotrophic mutant. Northern-blot analysis of purified Ade+ transformants demonstrated that both of the truncated mnp genes were regulated in a manner similar to the endogenous mnp genes with respect to nitrogen limitation and induction by Mn, HS, and H2O2.

Basidiomycota↗
PubMed1997

Long-term epilation using the EpiLight broad band, intense pulsed light hair removal system.

BACKGROUND: The long-term epilation of hair is the goal of several lasers and intense pulsed light systems. OBJECTIVE: The purpose of the study is to use the EpiLight Hair Removal System to assess long-term epilation and to assess its safety profile following a single treatment session. METHODS: Patients received a single treatment with the Epilight Hair Removal System after entering the patient's skin type, skin color, hair color, and hair density into the system's computer software. Treatment parameters include various wavelengths of light, pulse duration, pulse delay, and energy fluence. Thirty-seven subjects received a single treatment using one of four cut-off filters consisting of two to five pulses with energies of 34-55 J/cm2. RESULTS: The results of a single treatment show hair clearances occurring immediately and over a 12-week study period. Approximately 60% hair removal was noted at 12 weeks. CONCLUSIONS: The EpiLight Hair Removal System is an effective and safe method for long-term epilation of unwanted hair.

Adolescent↗
PubMed1996

Purification and characterization of two manganese peroxidase isozymes from the white-rot basidiomycete Dichomitus squalens.

Two manganese peroxidase isozymes, MnP1 and MnP2, were purified from the extracellular medium of ligninolytic cultures of Dichomitus squalens. The proteins were purified to homogeneity using DEAE-Sepharose chromatography and Mono Q fast protein liquid chromatography. MnP1 and MnP2 have molecular masses of 48000 and 48900 Da, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both isozymes are glycoproteins and each contains one iron protoporphyrin IX as a prosthetic group. The pl values of MnP1 and MnP2 are 4.15 and 3.90, respectively. N-Terminal amino-acid analysis suggests that these proteins are encoded by distinct genes. The Soret bands of the native ferric enzymes (408 nm and 406 nm, respectively) are shifted to 434 nm in the reduced enzymes and to 422 nm in the reduced-CO complexes. EPR g-values of the native enzymes are essentially identical to those for other MnPs and lignin peroxidases, and they confirm the high-spin state of the iron. The addition of 1 equivalent of H2O2 to either of the native ferric isozymes yields spectra which are characteristic of compound 1. Successive additions of 1 equivalent of ferrocyanide and 1 equivalent of H2O2 to the native enzymes yield spectra which are characteristic of compound II. Both MnP isozymes oxidize Mn2+ to Mn3+ in the presence of organic acid chelators. The MnP isozymes are produced by D. squalens only when the cells are grown in the presence of Mn.

Basidiomycota↗
PubMed1996

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.

Aspartic Acid↗
PubMed1996

1,4-Benzoquinone reductase from basidiomycete Phanerochaete chrysosporium: spectral and kinetic analysis.

The reaction mechanism of a 1,4-benzoquinone reductase from the wood-rotting basidiomycete Phanerochaete chrysosporium was investigated. The native, oxidized, FMN-containing enzyme was reduced quantitatively by NADH and the resulting reduced enzyme was reoxidized in the presence of one equivalent of 2,6-di-methoxy-1,4-benzoquinone (DMBQ). The stoichiometry of NADH oxidation versus DMBQ reduction is 1:1. The enzyme catalyzes the reduction of quinones to hydroquinones by a ping-pong steady-state mechanism. However, inhibition is observed at low NADH concentrations. Quinone products derived from the autooxidation of the unstable compounds 1,2,4-trihydroxybenzene and 5-chloro-2,3,4-trihydroxybenzene also appear to be substrates for the quinone reductase. The enzyme reduces the one-electron acceptors ferricyanide and ferricytochrome c (Cc3+) with rates of 58.4 and 0.08%, respectively, compared to DMBQ. The stoichiometry of NADH oxidation versus ferricyanide reduction is 1:2. In the presence of quinones the rates of Cc3+ and ferricyanide reduction are increased, owing to the nonenzymatic reduction of these acceptors by enzyme-generated hydroquinone products. Dicumarol and Cibacron blue are competitive inhibitors with respect to NADH, with Ki values of 2.1 and 0.30 microM, respectively. Reconstitution of the apoprotein with FMN yields a fully active enzyme at an FMN-to-protein ratio of 2:1, suggesting that the flavin content of the enzyme is two molecules of FMN per dimer.

Basidiomycota↗
PubMed1996

Oxidation of dimethoxylated aromatic compounds by lignin peroxidase from Phanerochaete chrysosporium.

The stabilities of the cation radicals of veratryl alcohol, 3,4-dimethoxytoluene and 1,4-dimethoxybenzene were compared by monitoring the formation of dimeric products during the oxidation of these substrates by lignin peroxidase (LiP). LiP oxidized veratryl alcohol to generate veratraldehyde as the major product. Several other monomeric products were obtained in low yield. Dimeric products resulting from the coupling of two cation radicals, or a cation radical with a neutral molecule, were obtained only in trace amounts or not at all. This suggests that the cation radical of veratryl alcohol rapidly loses a benzylic proton to form a benzylic radical which undergoes further reactions to form veratraldehyde. In contrast, the LiP oxidation of 3,4-dimethoxytoluene generated the dimeric product 3-(2,3-dimethoxy-6-methylphenyl)-4-methyl-1,2-benzoquinone as the major product. Several other monomeric and dimeric products were produced in lower yields. The generation of these dimeric products indicates that the cation radical of 3,4-dimethoxytoluene is considerably more stable than that of veratryl alcohol. This suggests that the electronegative benzylic oxygen of veratryl alcohol increases the acidity of the benzylic protons, destabilizing the veratryl alcohol cation radical. LiP oxidized 1,4-dimethoxybenzene to generate 1,4-benzoquinone and 2-(2,5-dimethoxyphenyl)-1,4-benzoquinone as the major products. The formation of these products indicates that the cation radical of 1,4-dimethoxybenzene also is relatively stable, as previously demonstrated by ESR. All of these results indicate that the veratryl alcohol cation radical generated by LiP oxidation is unstable, suggesting that it would not act as a diffusable radical mediator in LiP-catalyzed reactions.

Anisoles↗
PubMed1995

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)

Binding Sites↗
PubMed1995

Regulation of manganese peroxidase gene transcription by hydrogen peroxide, chemical stress, and molecular oxygen.

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of the lignin-degrading fungus Phanerochaete chrysosporium is regulated at the level of gene transcription by H2O2 and various chemicals, including ethanol, sodium arsenite, and 2,4-dichlorophenol, as well as by Mn(II) and heat shock. Northern (RNA) blot analysis demonstrates that the addition of 1.0 mM H2O2 to 5-day-old cultures grown in the absence of Mn results in the appearance of mnp mRNA within 15 min. Higher levels of mnp mRNA are obtained with simultaneous induction by Mn and H2O2 than with H2O2 alone. Although neither MnP activity nor associated protein is detectable in H2O2-induced cultures grown in the absence of Mn, simultaneous induction with Mn and H2O2 results in a 1.6-fold increase in MnP activity compared with the MnP activity resulting from Mn induction alone. In the presence of Mn, purging of low-nitrogen cultures with 100% O2, in contrast to incubation under air, results in an increase in the accumulation of mnp mRNA and a 13-fold increase in MnP activity on day 5. However, in contrast to the effects of H2O2 and heat shock, O2 purging of Mn-deficient cultures results in negligible accumulation of mnp mRNA.

Biodegradation, Environmental↗
PubMed1995

Fibrel.

Fibrel is a useful product for the treatment of depressed cutaneous scars, facial lines, and wrinkles. There have been documented increases in collagen production and inflammatory response over time. Adverse reactions seem to be minimal. If a more user-friendly product can be used--no plasma, minimal inflammatory response, 30-gauge needle--Fibrel should be considered in all patients for soft tissue augmentation.

Aminocaproates↗
PubMed1994

The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution.

The crystal structure of manganese peroxidase (MnP) from the lignin-degrading basidiomycetous fungus Phanerochaete chrysosporium has been solved using molecular replacement techniques and refined to R = 0.20 at 2.0 A. The overall structure is similar to that of two other fungal peroxidases, lignin peroxidase from P. chrysosporium and Arthromyces ramosus peroxidase. Like the other fungal peroxidases, MnP has two structural calcium ions. MnP also has two N-acetylglucosamine residues N-linked to Asn131 that are readily visible in the electron density map. The active site, consisting of a proximal His ligand H-bonded to an Asp residue and a distal side peroxide binding pocket consisting of a catalytic His and Arg, is the same as in the aforementioned fungal peroxidases as well as yeast cytochrome c peroxidase. MnP differs in having five rather than four disulfide bonds. The additional disulfide bond, Cys341-Cys348, is located near the C terminus of the polypeptide chain. Importantly, a new cation binding site, which we propose is the manganese-binding site of MnP, was located in the crystal structure. The ligands constituting the Mn(2+)-binding site include Asp179, Glu35, Glu39, a heme propionate, and two water molecules. Electron transfer from Mn2+ to the heme edge or iron center is envisioned to occur through a sigma-bonded pathway along a heme propionate.

Amino Acid Sequence↗
PubMed1994

Aromatic nitroreductase from the basidiomycete Phanerochaete chrysosporium.

A membrane-associated aromatic nitroreductase activity was identified in cell-free extracts of the lignin-degrading fungus Phanerochaete chrysosporium. The enzyme catalyzed the nitro group reduction of 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, 1-chloro-2,4-dinitrobenzene, and 2,4-dichloro-1-nitrobenzene. The corresponding hydroxylamines and/or amines were identified as reaction products by HPLC and/or GC-MS. 1-Nitroso-3-nitrobenzene and 1-hydroxylamino-3-nitrobenzene also were reduced by the enzyme, suggesting they were intermediates in the reaction. The enzyme required NAD(P)H as a cosubstrate and the optimal pH and temperature for the reaction were 6.5 and 50 degrees C, respectively. Enzyme activity was not observed in the presence of molecular oxygen. The membrane-associated enzyme could be solubilized with the nonionic detergent Triton X-100.

Basidiomycota↗
PubMed1994

Oxidation of dibenzo-p-dioxin by lignin peroxidase from the basidiomycete Phanerochaete chrysosporium.

Dibenzo-p-dioxin (I) was rapidly degraded in ligninolytic cultures of the basidiomycete Phanerochaete chrysosporium. Lignin peroxidase (LiP) oxidized I to generate the following products: catechol (V), dibenzo-p-dioxin-2,3-quinone (VIII), 2-hydroxy-5-(2-hydroxyphenoxy)-1,4-benzoquinone (IX), 4,5-dihydroxy-1,2-benzoquinone (X), 2-(2-hydroxyphenoxy)-1,4-benzoquinone (XI), 4-hydroxy-1,2-benzoquinone (XII), and 1,2-benzoquinone (XIII). Identical products were formed when the reaction was conducted under argon. No incorporation of 18O into products was observed when the reaction was conducted under 18O2. Oxidation of I in H(2)18O resulted in incorporation of two atoms of 18O into the quinone VIII. Nonenzymatic hydrolysis of the quinone (VIII) yielded catechol (V), IX and X. Hydrolysis of VIII in H(2)18O resulted in incorporation of 18O atoms into IX and X, whereas no incorporation of 18O atoms into V was observed. These results are explained by mechanisms involving the one-electron oxidation of I by LiP to produce the corresponding cation radical. Nucleophilic attack of water on the cation radical generates a 2-hydroxydibenzo-p-dioxin radical, which is oxidized to a delocalized cation. The attack of water at position C-4a of the 2-hydroxydibenzo-p-dioxin cation, followed by oxidation and C-O-C bond cleavage, lead to formation of the quinone (XI), which undergoes 1,4-addition of water and cleavage of the second C-O-C bond to generate V and XII.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗
PubMed1994

Mechanism of manganese peroxidase compound II reduction. Effect of organic acid chelators and pH.

The effect of oxalate, malonate, lactate, and succinate chelators on the reduction of Phanerochaete chrysosporium manganese peroxidase compound II by MnII was investigated using stopped-flow techniques. All rate data were collected from single-turnover experiments under pseudo-first-order conditions. With oxalate, the reduction of compound II by MnII exhibited saturation behavior when the observed pseudo-first-order rate constants were plotted against oxalate concentration. The plots passed through the origin, indicating that the reduction by MnII is irreversible at all concentrations of oxalate. Maximal stimulation of the rate of compound II reduction occurred at 2 mM oxalate, the concentration of oxalate found in the extracellular medium of agitated cultures of this fungus. In contrast, maximal stimulation of the reduction of compound II by MnII only was observed at high (> 20 mM) nonphysiological concentrations of malonate and lactate. Furthermore, at low concentrations of malonate and lactate, the reduction of compound II appeared to be reversible. These results suggest that at physiological concentrations oxalate chelates and stabilizes MnIII, enhancing its efficient removal from the enzyme. The rate constants for compound II reduction exhibited bell-shaped curves as a function of pH and had optima at pHs 5.0-5.4. In the presence of succinate, triphasic kinetics were observed for compound II reduction by MnII. In contrast to the reduction of compound II by MnII, various chelators had no observable effect on the formation of compound I. However, they did affect the steady-state oxidation of 2,6-dimethoxyphenol.

Basidiomycota↗