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RationaleEffective analytical techniques are needed to characterize lignin products for the generation of renewable carbon sources. Application of matrix‐assisted laser desorption/ionization (MALDI) in lignin analysis is limited because of poor ionization efficiency. In this study, we explored the potential of cationization along with a 2,5‐dihydroxyacetophenone (DHAP) matrix to characterize model lignin oligomers. MethodsSynthesized lignin oligomers were analyzed using the developed MALDI method. Two matrix systems, DHAP and α‐cyano‐4‐hydroxycinnamic acid (CHCA), and three cations (lithium, sodium, silver) were evaluated using a Bruker UltraFlextreme time‐of‐flight mass spectrometer. Instrumental parameters, cation concentration, matrix, sample concentrations, and sample spotting protocols were optimized for improved results. ResultsThe DHAP/Li+combination was effective for dimer analysis as lithium adducts. Spectra from DHP and ferric chloride oligomers showed improved signal intensities up to decamers (m/z1823 for the FeCl3system) and provided insights into differences in the oligomerization mechanism. Spectra from a mixed DHP oligomer system containing H, G, and S units showed contributions from all monolignols within an oligomer level (e.g. tetramer level). ConclusionsThe DHAP/Li+method presented in this work shows promise to be an effective analytical tool for lignin analysis by MALDI and may provide a tool to assess lignin break‐down efforts facilitating renewable products from lignin.
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Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation
Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism oferythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported inNovosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that convertserythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of theN. aromaticivoransLdpA and another homolog identified inSphingobiumsp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1reaction product, and we further demonstrated that both enantiomers oferythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
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- Award ID(s):
- 2153972
- PAR ID:
- 10505405
- Publisher / Repository:
- National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 120
- Issue:
- 4
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2212246120
