Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase

Smith, Andrew T.; Doyle, Wendy A.; Dorlet, Pierre; Ivancich, Anabella

doi:10.1073/pnas.0904535106

Cited by 77 publications

(101 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…; Table S5) (16)(17)(18)(19)(20)(21). A similar g-tensor anisotropy effect would be expected for a hydroxylated Trp radical because the radical spin density is typically delocalized over the entire indole ring (22)(23)(24)(25)(26)(27).…”

Section: Resultsmentioning

confidence: 53%

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Yukl

Liu

Krzystek

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual sixelectron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.cofactor biosynthesis | tryptophan radical | heme | posttranslational modification | electron transfer P rotein-based radicals, particularly on tyrosine (Tyr) and tryptophan (Trp) residues, have been implicated in a large number of catalytic and electron transfer reactions in biology (1), including the long-range electron transfer reactions required for photosynthesis (2), respiration (3), and DNA synthesis (4) and repair (5). Aberrant formation of protein radicals during oxidative stress is also of special importance. Evidence for the involvement of protein-based and substrate-based radicals in enzyme-catalyzed reactions has increased substantially in recent years, and expanded our knowledge of the scope of chemical reactions accessible to enzymes. The ability of enzymes to catalyze what were previously thought to be unattainable reactions is exemplified by MauG. The biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH) requires posttranslational modification of two tryptophan residues. MADH from Paracoccus denitrificans is a 119-kDa α 2 β 2 heterotetramer that contains two active sites and two TTQ cofactors derived from residues βTrp57 and βTrp108 (6). MauG is a c-type diheme enzyme that catalyzes the conversion of a MADH precursor (preMADH) that has one oxygen atom already inserted into the βTrp57 indole ring (βTrp57-OH of preTTQ) (7), to mature TTQ-containing MADH (8) (Fig. 1).Catalysis by MauG proceeds via a bis-Fe(IV) redox state, which may be generated by reaction of di-Fe(II) MauG with O 2 or di-Fe(III) MauG with H 2 O 2 (9). Catalytically active crystals of the MauG-preMADH protein complex show that the site of TTQ formation on β-preMADH is 40.1 Å from the MauG highspin heme iron...

show abstract

Section: Resultsmentioning

confidence: 53%

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Yukl

Liu

Krzystek

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…However, unlike R. jostii DyPB and P. fluorescens DyP1B, TfuDyP shows no activity for Mn 2+ oxidation, hence the lignin oxidation activity of TfuDyP must be due either to direct oxidation by the heme cofactor, or electron transfer via a surface redox-active residue, as is known for fungal lignin peroxidase [28]. The lack of Mn 2+ oxidation activity has also been observed in other A-type DyP enzymes [7,15].…”

Section: Discussionmentioning

confidence: 74%

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds

Rahmanpour

Rea

Jamshidi

et al. 2016

Archives of Biochemistry and Biophysics

105

View full text Add to dashboard Cite

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at max 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a -aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS.

show abstract

“…The hydroxyl group Tyr-337 forms a strong hydrogen bond of 2.6 Å with the carboxylate of Glu-354. It has been reported that acidic side chains in the vicinity of a tryptophan or a tyrosine stabilize an emerging radical cation (26,47,48). Alignments with other DyPs show that Tyr-337 is a conserved residue in fungal as well as bacterial DyPs and the related enzymes TyrA from Shewanella oneidensis and EfeB from Escherichia coli but is missing in Cld-like proteins (5).…”

Section: Resultsmentioning

confidence: 99%

First Crystal Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase

Strittmatter

Liers

Ullrich

et al. 2013

Journal of Biological Chemistry

116

View full text Add to dashboard Cite

Background: DyP-type peroxidases catalyze biotechnologically important reactions. Results: Based on the crystal structure of a fungal DyP, the conformational flexibility of Asp-168 is elucidated. Tyr-337 is identified as a surface-exposed substrate interaction site. Conclusion: Asp-168 and Tyr-337 are key residues directly involved in AauDyPI-catalysis. Significance: Peroxidases are biocatalysts, much sought after and ubiquitous enzymes in nature.

show abstract

Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase

Abstract: heme peroxidase ͉ high-field EPR spectroscopy ͉ protein engineering ͉ tryptophan radical ͉ electron transfer

Cited by 77 publications

References 35 publications

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds

First Crystal Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase

Contact Info

Product

Resources

About