It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and recombinant LiP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be implicated in catalysis. The role of this residue has therefore been examined using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectively). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using 1H NMR spectroscopy and its integrity confirmed prior to the kinetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the "classical heme edge" substrate access channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8), showed substantial activity with respect to veratryl alcohol (VA) oxidation and a marked (2.4 pH units) increase in pKa for the oxidation of a negatively charged difluoroazo dye. More surprisingly, the Trp171 LiPH8 mutants W171F and W171S LiPH8 were found to have lost all activity with VA as substrate, and compounds I and II were unable to react with VA. Both mutants, however, retained substantial activity with two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a heme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the oxidation of VA. Stopped-flow kinetic studies showed that all the mutants examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s-1), typical of wild-type LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer pathway must exist within the protein leading from the heme to a surface site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171.
In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group. To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide. Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan. At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda max = 333 nm can be followed that is identical to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan. The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature. However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171. Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification. We conclude that the hydroxylation at the C beta of Trp171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P. chrysosporium, which are known to contain an oxidase-based H2O2-generating system. No dependence on dioxygen was found for this oxidative process. Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced activity with respect to the substrate veratryl alcohol. This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions.
The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1. 4.3.3) from Trigonopsis variabilis with [␣-1 H]-and [␣-2 H]phenylglycine has been determined. The pH dependence of V max is compatible with pK a values of Ϸ8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is Ϸ3.9, and the solvent isotope effect Ϸ1.6. The reductive halfreaction is biphasic, the first, fast phase, k 2 , corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k ؊2 , the reversal of k 2 . D-Amino acid oxidase (EC 1.4.3.3, DAAO) 1 is the paradigm of flavin enzymes. It was the second flavoprotein to be uncovered, and probably it is the most studied member of this superfamily. In addition to the classical protein from mammalian kidney, recently related DAAOs have been described from various yeasts (1, 2). A common feature of all these enzymes is the dehydrogenation of D-amino acids to yield ␣-imino and, upon subsequent hydrolysis, ␣-ketoacids. The terminal redox acceptor is dioxygen. In spite of the innumerable studies, the molecular mechanism by which this enzyme brings about substrate dehydrogenation is far from being solved. Mechanistic proposals revolve around possible modes by which the substrate ␣C-H bond is being broken in the step critical for catalysis.The most prominent proposal is the carbanion mechanism, which is characterized by initial abstraction of the ␣-H as H ϩ leading to an intermediate in which the ␣-carbon carries a negative charge. Evidence in its favor has been discussed in various review articles (3, 4). So called "hydride mechanisms" in which a H Ϫ is expulsed from ␣C-H also have been discussed at various occasions but have not been proposed explicitly until most recently by Mattevi et al. (5). From Miura and Miyake (6) stems a proposal in which "the lone-pair electrons of the neutral amino group of the substrate are transferred to the flavin in a concerted manner with the abstraction of the ␣-proton." (For schematic representations of the mechanisms and structures see Denu and Fitzpatrick (7).) An approach to investigate the molecular mechanisms of enzymes consists in the correlation of reactivities (reaction rates) with the properties of substrate substituents which influence the steric or electronic properties of the latter. This approach was advocated originally by Hammett (8) for chemical systems and was extended by Hansch and Leo (9). Klinman and co-workers (10, 11) have pioneered its use in the study of enzymatic reactions. Recently Walker and Edmondson (12) have used it to study monoamine oxidase. As early as in 1966 Neims et al. (13) have employed substituted phenylglycines for probing the mechanism of pkDAAO; however, the results were contradictory. In retrospect the reason for this is clear: these authors did rely on the correlations of V max da...
Lignin peroxidase (LiP) plays a central role in the biodegradation of the plant cell wall constituent lignin. LiP is able to oxidize aromatic compounds with redox potentials higher than 1.4 V (NHE) by single electron abstraction, but the exact redox mechanism is still poorly understood. The finding in our laboratory that the Cβ-atom of Trp171 carries a unique modification led us to initiate experiments to investigate the role of this residue. These experiments, employing crystallography, site-directed mutagenesis, protein chemistry, spin-trapping and spectroscopy, yielded the following results: (i) Trp171 is stereospecifically hydroxylated at its Cβ-atom as the result of an auto-catalytic process, which occurs under turnover conditions in the presence of hydrogen peroxide, (ii) Evidence for the formation of a Trp171 radical intermediate has been obtained using spin-trapping, in combination with peptide mapping and protein crystallography. (iii) Trp171 is very likely to be involved in electron transfer from natural substrates to the haem cofactor via LRET. (iv) Mutagenetic substitution of Trp171 abolishes completely the oxidation activity for veratryl alcohol, but not for artificial substrates. (v) Structural changes in response to the mutation are marginal. Therefore the lack of activity is due to the absence of the redox active indole side chain.
Lignin peroxidase (LiP) plays a central role in the biodegradation of the plant cell wall constituent lignin. LiP is able to oxidize aromatic compounds with redox potentials higher than 1.4 V (NHE) by single electron abstraction, but the exact redox mechanism is still poorly understood. The finding in our laboratory that the Cbeta-atom of Trp171 carries a unique modification led us to initiate experiments to investigate the role of this residue. These experiments, employing crystallography, site-directed mutagenesis, protein chemistry, spin-trapping and spectroscopy, yielded the following results: (i) Trp171 is stereospecifically hydroxylated at its Cbeta-atom as the result of an auto-catalytic process, which occurs under turnover conditions in the presence of hydrogen peroxide. (ii) Evidence for the formation of a Trp171 radical intermediate has been obtained using spin-trapping, in combination with peptide mapping and protein crystallography. (iii) Trp171 is very likely to be involved in electron transfer from natural substrates to the haem cofactor via LRET. (iv) Mutagenetic substitution of Trp171 abolishes completely the oxidation activity for veratryl alcohol, but not for artificial substrates. (v) Structural changes in response to the mutation are marginal. Therefore the lack of activity is due to the absence of the redox active indole side chain.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.