Hydrogen peroxide
is a cosubstrate for the oxidative cleavage of
saccharidic substrates by copper-containing lytic polysaccharide monooxygenases
(LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high,
but it is accompanied by rapid inactivation of the enzymes, presumably
through protein oxidation. Herein, we use UV–vis, CD, XAS,
EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with
mass spectrometry and DFT calculations, to show that the product of
reaction of an AA9 LPMO with H2O2 at higher
pHs is a singlet Cu(II)–tyrosyl radical species, which is inactive
for the oxidation of saccharidic substrates. The Cu(II)–tyrosyl
radical center entails the formation of significant Cu(II)–(●OTyr) overlap, which in turn requires that the plane
of the d(x
2–y
2) SOMO of the Cu(II) is orientated toward the tyrosyl radical.
We propose from the Marcus cross-relation that the active site tyrosine
is part of a “hole-hopping” charge-transfer mechanism
formed of a pathway of conserved tyrosine and tryptophan residues,
which can protect the protein active site from inactivation during
uncoupled turnover.
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Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2’-phosphate being pushed inside the NADP
+
binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.
Among the molecular mechanisms of adaptation in biology, enzyme functional diversification is indispensable. By allowing organisms to expand their catalytic repertoires and adopt fundamentally different chemistries, animals can harness or eliminate new-found substances and xenobiotics that they are exposed to in new environments. Here, we explore the flavin-containing monooxygenases (FMOs) that are essential for xenobiotic detoxification. Employing a paleobiochemistry approach in combination with enzymology techniques we disclose the set of historical substitutions responsible for the family’s functional diversification in tetrapods. Remarkably, a few amino acid replacements differentiate an ancestral multi-tasking FMO into a more specialized monooxygenase by modulating the oxygenating flavin intermediate. Our findings substantiate an ongoing premise that enzymatic function hinges on a subset of residues that is not limited to the active site core.
In the version of this article initially published, in Fig. 6b, top left panel, the β-sheet was labeled as a "Three-stranded bridging sheet. " The correct label is "Four-stranded bridging sheet. " The error has been corrected in the HTML and PDF versions of the article.
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