Biomedical subjects

M Tien

Publications and source records attributed to M Tien.

At least 19 recordsLinked to original sources

PubMed2001

Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium.

A Mn(2+)-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn(2+) oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn(2+) and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The K(m) for Mn(2+) was determined to be 1.57 mM and the k(cat) = 5.45 s(-1). These results indicate that the mutated lignin peroxidase is less effective in Mn(2+) oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.

Amino Acid Sequence↗

PubMed2001

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Amino Acid Sequence↗

PubMed2001

Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase.

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.

Base Sequence↗

PubMed2000

Reactivity of manganese peroxidase: site-directed mutagenesis of residues in proximity to the porphyrin ring.

The purpose of this study was to determine the effect of heme pocket hydrophobicity on the reactivity of manganese peroxidase. Residues within 5 A of the heme active site were identified. From this group, Leu169 and Ser172 were selected and mutated to Phe and Ala, respectively. The mutant proteins were then characterized by steady-state kinetics. Whereas the Leu169Phe mutation had little, if any, effect on activity, the Ser172Ala mutation decreased kcat and also the specificity constant (kcat/Km) for Mn2+, but not H2O2. Transient-state studies indicated that the mutation affected only the reactions of compound II. These results indicate that compound II is the most sensitive to changes in the heme environment.

Alanine↗

PubMed2000

Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket.

Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an 'alkaline' and an 'acid' transition), which indicates that (at least) two protonating groups change their pK(b) values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His(173), in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N(delta) of the proximal imidazole group is redox-linked and that it is crucial in regulating the 'alkaline' transition. On the other hand, none of the proximal mutants alters the 'acid' transition, suggesting that it is modulated by groups located in a different portion of the protein.

Electrochemistry↗

PubMed1999

Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium.

Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H(2)O(2) were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn(2+) from 233 s(-1) (wild type) to 154 s(-1) and 107 s(-1), respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn(2+) to 320 s(-1). The F190L mutation decreased this rate to 137 s(-1). The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn(2+) from 140 s(-1) in the wild type to 36 s(-1). There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe(3+)/Fe(2+) couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heme and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.

Amino Acid Substitution↗

PubMed1999

Spectroscopic characterization of active mutants of manganese peroxidase: mutations on the proximal side affect calcium binding of the distal side.

The mutants at position 242 of manganese peroxidase (MnP), where the native Asp has been substituted with a Ser or a Glu, have been shown to be active, and are here characterized by electronic, EPR, and NMR spectroscopies. We have also mutated another residue on the proximal side, Phe 190 to Val and Leu, yielding active mutants. When studied by the above-mentioned spectroscopies, the mutants at both positions 242 and 190 exhibit three pH-dependent transitions. In contrast to the transitions observed at low and high pH, the spectroscopic studies reveal that the transition at intermediate pH has pK(a) values up to 2 units lower for the mutants at D242E and -S and F190V than for the wild type. This process is due to the ionization of a group that affects the transition to the bis-histidine coordination at the iron. The observed changes in the pK(a) values are related to the altered affinity of the calcium-binding site in the distal pocket. Other variations are observed in the other two pK(a) values. Characterization of the cyanide derivatives indicates that the location and orientation of the distal and proximal His residues are essentially identical to that in the wild type. Our results indicate that mutations on the proximal side residues can affect changes in the distal side. In particular, deprotonation of a group, whose pK(a) is influenced by the nature of the residues in the proximal side, produces a movement of helix B, which in turn induces the coordination of the distal His and the loss of the distal calcium ion.

Aspartic Acid↗

PubMed1999

Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation.

The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (GS) and BSA is greatly influenced by pH and CO2. At physiological concentrations of CO2 (1.3 mM), the generation of carbonyl groups (0.2-0.4 equivalents/subunit) is little affected by pH over the range of 7.2-9.0, but, in the absence of CO2, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH. Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives. In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO2. However, the pH profiles of tyrosine nitration in GS and BSA are not the same. With both proteins, nitration decreases approximately 65% with increasing pH over the range of 7.2-8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4-8.8. The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at both high and low pH values. These results emphasize the importance of controlling the pH and CO2 concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO2 concentrations.

Carbon Dioxide↗

PubMed1999

Myeloperoxidase-catalyzed oxidation of tyrosine.

The oxidation of tyr by myleoperoxidase (MPO) is postulated to play a role in atherosclerotic plaque formation. MPO has been localized in plaques and a product of MPO-catalyzed oxidation of tyr, dityrosine, also found in plaques, is proposed to be a protein cross-linking agent. We have performed kinetic studies on the oxidation of tyr by MPO and investigated the role of substrate size on its oxidation. The kinetics of MPO-catalyzed oxidation of tyr where the tyr is free tyr, the dipeptides, tripeptides, and polypeptides were studied by stopped-flow methods. The rate of reaction with enzyme intermediates compound I and compound II are decreased with increasing substrate size. The amount of dityrosine formed was also decreased with increasing substrate size. The ability of sulfhydryl compounds to inhibit MPO-dependent dityrosine formation was investigated with reduced glutathione, cys, and met. Glutathione and cys both served as substrates for MPO compound I but not compound II, whereas met was not a substrate for either compound I or II. Met, an amino acid postulated to act as a "last chance" antioxidant for proteins, was not able to inhibit dityrosine formation from MPO-catalyzed oxidation of tyr. Glutathione and cys caused partial inhibition; however, it is possible that this inhibition was due to their ability to react directly with MPO rather than trapping the tyr radicals.

Animals↗

PubMed1999

Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers.

The oxidation of phenolic oligomers by lignin and manganese peroxidases was studied by transient-state kinetic methods. The reactivity of peroxidase intermediates compound I and compound II was studied with the phenol guaiacol along with a beta-O-4 phenolic dimer, trimer, and tetramer. Compound I of both peroxidases is much more reactive than compound II. The rate constants for these substrates with Mn peroxidase compound I range from 1.0 x 10(5) M-1 s-1 for guaiacol to 1.1 x 10(3) M-1 s-1 for the tetramer. Reactivity is much higher with lignin peroxidase compound I with rate constants ranging from 1.2 x 10(6) M-1s-1 for guaiacol to 3.6 x 10(5) M-1 s-1 for the tetramer. Rate constants with compound II are much lower with Mn peroxidase exhibiting very little reactivity. The rate constants dramatically decreased with both peroxidases as the size of the substrate increased. The extent of the decrease was much more dramatic with Mn peroxidase, leading us to conclude that, despite its ability to oxidize phenols, Mn2+ is the only physiologically significant substrate. The rate decrease associated with increasing substrate size was more gradual with lignin peroxidase. These data indicate that whereas Mn peroxidase cannot efficiently directly oxidize the lignin polymer, lignin peroxidase is well suited for direct oxidation of polymeric lignin.

Catalysis↗

PubMed1998

Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis.

Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.

Alanine↗

PubMed1998

Monitoring the role of oxalate in manganese peroxidase.

The water proton relaxation rate measurements between 0.01 and 50 MHz on water solutions containing the cyanide adduct of the manganese-depleted manganese peroxidase (MnP-CN-) and increasing amounts of Mn2+ have been determined. The proton relaxivity curves have shown evidence of the formation of the protein/Mn2+ complex and have been analyzed in order to obtain spin Hamiltonian parameters and correlation times. Oxalate is shown not to alter the above profiles. This suggests that no protein-Mn2+-oxalate ternary complex is formed and that oxalate does not remove Mn2+ from the protein. On the basis of high-resolution 1H NMR experiments, we propose that Ce3+ and Gd3+ bind at the manganese site, and, on the basis of the charge, we propose that they may mimic Mn3+. The water proton relaxation rates of water solutions containing manganese-depleted MnP-CN- and increasing amounts of Gd3+ have been measured and analyzed. Oxalate is shown to remove the trivalent metal ions. This suggests that trivalent metal ions bind oxalate and diffuse away from the protein presumably as oxalate complexes. Implications for the enzymatic mechanism are discussed.

Binding Sites↗

PubMed1998

Cloning of Phanerochaete chrysosporium leu2 by complementation of bacterial auxotrophs and transformation of fungal auxotrophs.

A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, lambda YES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the beta-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.

3-Isopropylmalate Dehydrogenase↗

PubMed1997

Mutagenesis of the Mn2+-binding site of manganese peroxidase affects oxidation of Mn2+ by both compound I and compound II.

The present study investigates whether compound I and compound II of manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium utilize the same Mn-binding site for catalysis. Manganese peroxidase was expressed from its cDNA in Escherichia coli and refolded from inclusion bodies to yield fully active enzyme. Three mutants of the enzyme were generated by site-directed mutagenesis. Each of the three amino acid residues proposed to be involved in Mn2+ binding, E35, D179, and E39, was mutated. The acidic side chains of E35 and E39 were shortened by one carbon to the acidic group D, and the acidic side chain of D179 was shortened by one carbon to the alkyl group A. These mutants, E35D, D179A, and E39D, were used to determine whether Mn2+ reacts at the same site with both compound I and compound II of manganese peroxidase and to determine whether phenolic substrates for the enzyme react at this site. Our results conclusively demonstrate that E35 and D179 residues are involved not only in Mn2+ binding but also in electron transfer from Mn2+ to the enzyme for both compound I and compound II. In contrast, E39 is not critically important to either process. None of the three residues is involved in reactions with phenolic substrates or with H2O2.

Basidiomycota↗

PubMed1997

Oxidation of 4-methoxymandelic acid by lignin peroxidase. Mediation by veratryl alcohol.

The mechanism of veratryl alcohol-mediated oxidation of 4-methoxymandelic acid by lignin peroxidase was studied by kinetic methods. For monomethoxylated substrates not directly oxidized by lignin peroxidase, veratryl alcohol has been proposed to act as a redox mediator. Our previous study showed that stimulation of anisyl alcohol oxidation by veratryl alcohol was not due to mediation but rather due to the requirement of veratryl alcohol to complete the catalytic cycle. Anisyl alcohol can react with compound I but not with compound II. In contrast, veratryl alcohol readily reduces compound II. We demonstrate in the present report that the oxidation of 4-methoxy mandelic acid is mediated by veratryl alcohol. Increasing veratryl alcohol concentration in the presence of 2 mM 4-methoxymandelic acid resulted in increased oxidation of 4-methoxymandelic acid yielding anisaldehyde. This is in contrast to results obtained with anisyl alcohol where increased concentrations of veratryl alcohol caused a decrease in product formation. ESR spectroscopy demonstrated that 4-methoxymandelic acid caused a decrease in the enzyme-bound veratryl alcohol cation radical signal, which is consistent with its reaction at the active site of the enzyme.

Benzyl Alcohols↗

PubMed1997

Role of calcium in maintaining the heme environment of manganese peroxidase.

We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland & Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pKa of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near-ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

Basidiomycota↗

PubMed1996

Heterologous expression and reconstitution of fungal Mn peroxidase.

We have optimized the conditions under which recombinant Mn peroxidase from the white-rot fungus Phanerochaete chrysosporium can be expressed in Escherichia coli. A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (lambda MP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 1013-1017, 1995) whose expression in E. coli results in the formation of catalytically inactive polypeptide which can be refolded to active enzyme. The refolded enzyme was purified to homogeneity. Refolding was most efficient in 2 M urea, pH 8.0, and was absolutely dependent upon the presence of CaCl2, hemin, and oxidized glutathione. The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme. The Km of recombinant Mn peroxidase for substrates H2O2 and the Mn2+/oxalate complex are 100 and 52 microM, respectively. The kcat as measured by Mn3+/oxalate formation is 450 s-1. These are essentially the same values as seen with the native fungal enzyme. The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 x 10(6) M-1 s-1, identical to the rate of the native fungal Mn peroxidase. The reaction of compound I with Mn2+ is too fast to measure at pH 4.5 in the recombinant Mn peroxidase. At a suboptimal pH of 2.5 a rate of 4.2 x 10(4) M-1 s-1 is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has a Kd of 13 microM and a first-order rate constant of 230 s-1 in the recombinant enzyme. These rates are essentially the same as those seen with the native fungal MnP. These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase.

Agaricales↗

PubMed1996

Oxidation of 1,2,4,5-tetramethoxybenzene by lignin peroxidase of Phanerochaete chrysosporium.

We have reinvestigated the lignin peroxidase-catalyzed oxidation of 1,2,4,5-tetramethoxybenzene (TMB) by using presteady-state and steady-state kinetic methods. Our presteady-state kinetic results show that the reaction of compound I with TMB obeyed second order kinetics with a rate constant of 1.1 x 10(7) M-1s-1. The reaction of compound II with TMB exhibits a hyperbolic concentration dependence with a Kd of 16 microM and K = 24 s-1. The stoichiometry of TMB oxidation during steady state is two TMB cation radicals formed per H2O2 consumed. These results clearly show that TMB is a good substrate for both compounds I and II of lignin peroxidase.

Anisoles↗