Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6

Kuatsjah, Eugene; Chan, Anson; Katahira, Rui; Haugen, Stefan J.; Beckham, Gregg T.; Murphy, M.E.P.; Eltis, Lindsay D.

doi:10.1016/j.jbc.2021.100758

Cited by 10 publications

(7 citation statements)

References 44 publications

(103 reference statements)

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“…Product inhibition of LdpA by lignostilbene may be an in vitro artifact that is not relevant in vivo as both N. aromaticivorans and SYK-6 encode multiple copies of lsd , many possessing cross-reactivities to different 4-hydroxystilbenes ( 19 , 20 , 30 – 33 ). The observed competitive product inhibition can readily be rationalized by the DFT calculation where the energy level associated with the erythro -DGPD and lignostilbene binding is comparable ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The threo -DGPD diastereomer, conversely, is not converted by this enzyme pair and its catabolic pathway has not yet been reported to our knowledge. Here, we propose a renaming of the γ-formaldehyde lyase to LdpA ( l ignin d iaryl p ropane-associated catabolic enzyme), formerly known as LsdE, in the light of eight stilbene dioxygenases characterized in SYK-6, which are also referred to as Lsd enzymes ( 19 , 20 ). The mechanism of the reaction catalyzed by LdpA was heretofore unknown, including the identity of the C 1 reaction product.…”

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confidence: 99%

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Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Kuatsjah

Zahn

Chen

et al. 2023

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

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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 of erythro -1,2-diguaiacylpropane-1,3-diol ( erythro -DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium 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 converts erythro -DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C 1 reaction product, and we further demonstrated that both enantiomers of erythro -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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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Kuatsjah

Zahn

Chen

et al. 2023

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

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“…Notably, choline dehydrogenase (betA) exhibited 40% of similarity to phcC/D in Sphingobium sp. SYK-6 based on blastp analysis in NCBI for P. putida KT2440 [ 44 , 49 , 50 ]. Proteomics results demonstrated the aldehyde dehydrogenase (aldA/aldB-I) presented in P. putida secretome (Additional file 2 : Table S4).…”

Section: Resultsmentioning

confidence: 99%

“…SYK-6 (Fig. 5 ) [ 44 , 49 , 50 ]. In addition, the presence of H 2 O 2 only slightly increased the β -5 linkage compared to secretome alone, suggesting the peroxidase might not involve in β -5 cleavage.…”

Section: Resultsmentioning

confidence: 99%

Understanding of bacterial lignin extracellular degradation mechanisms by Pseudomonas putida KT2440 via secretomic analysis

Peng

Kitata

et al. 2022

Biotechnol Biofuels

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Background Bacterial lignin degradation is believed to be primarily achieved by a secreted enzyme system. Effects of such extracellular enzyme systems on lignin structural changes and degradation pathways are still not clearly understood, which remains as a bottleneck in the bacterial lignin bioconversion process. Results This study investigated lignin degradation using an isolated secretome secreted by Pseudomonas putida KT2440 that grew on glucose as the only carbon source. Enzyme assays revealed that the secretome harbored oxidase and peroxidase/Mn2+-peroxidase capacity and reached the highest activity at 120 h of the fermentation time. The degradation rate of alkali lignin was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidase/Mn2+-peroxidase. Gas chromatography–mass spectrometry (GC–MS) and two-dimensional 1H–13C heteronuclear single-quantum coherence (HSQC) NMR analysis revealed that the oxidases exhibited strong C–C bond (β-β, β-5, and β-1) cleavage. The activation of peroxidases enhanced lignin degradation by stimulating C–O bond (β-O-4) cleavage, resulting in increased yields of aromatic monomers and dimers. Further mass spectrometry-based quantitative proteomics measurements comprehensively identified different groups of enzymes particularly oxidoreductases in P. putida secretome, including reductases, peroxidases, monooxygenases, dioxygenases, oxidases, and dehydrogenases, potentially contributed to the lignin degradation process. Conclusions Overall, we discovered that bacterial extracellular degradation of alkali lignin to vanillin, vanillic acid, and other lignin-derived aromatics involved a series of oxidative cleavage, catalyzed by active DyP-type peroxidase, multicopper oxidase, and other accessory enzymes. These results will guide further metabolic engineering design to improve the efficiency of lignin bioconversion. Graphical Abstract

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“…Notably, choline dehydrogenase (betA) exhibited 40% of similarity to phcC/D in Sphingobium sp. SYK-6 based on blastp analysis in NCBI for P.putida KT2440 [44,49,50]. Proteomics results demonstrated the aldehyde dehydrogenase (aldA/aldB-I) presented in P. putida secretome (Table S4).…”

Section: Gc-ms Analysis Of Lignin Breakdown Productsmentioning

confidence: 99%

Understanding of Bacterial Lignin Extracellular Degradation Mechanisms by Pseudomonas putida KT2440 via Secretomic Analysis

Peng

Kitata

et al. 2022

Preprint

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Background: Bacterial lignin degradation is believed to be primarily achieved by a secreted enzyme system. Effects of such extracellular enzyme systems on lignin structural changes and degradation pathways are still not clearly understood, which remains as a bottleneck in the bacterial lignin bioconversion process. Results: This study investigated lignin degradation using an isolated secretome secreted by Pseudomonas putida KT2440 that grew on glucose as the only carbon source. Enzyme assays revealed that the secretome harbored oxidase and peroxidase/Mn2+-peroxidase capacity and reached the highest activity at 120 h of the fermentation time. The degradation rate of alkali lignin was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidase/Mn2+-peroxidase. Gas chromatography-mass spectrometry (GC-MS) and two-dimensional 1H-13C heteronuclear single-quantum coherence (HSQC) NMR analysis revealed that the oxidases exhibited strong C-C bond (β-β, β-5, and β-1) cleavage. The activation of peroxidases enhanced lignin degradation by stimulating C-O bond (β-O-4) cleavage, resulting in increased yields of aromatic monomers and dimers. Further mass spectrometry-based quantitative proteomics measurements comprehensively identified different groups of enzymes particularly oxidoreductases in P. putida secretome, including reductases, peroxidases, monooxygenases, dioxygenases, oxidases, and dehydrogenases, potentially contributed to the lignin degradation process. Conclusions: Overall, we discovered that bacterial extracellular degradation of alkali lignin to vanillin, vanillic acid, and other lignin derived-aromatics involved a series of oxidative cleavage, catalyzed by active DyP-type peroxidase, multicopper oxidase, and other accessory enzymes. These resutls will guide further metabolic engineering design to improve the efficiency of lignin bioconversion.

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Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6

Cited by 10 publications

References 44 publications

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Understanding of bacterial lignin extracellular degradation mechanisms by Pseudomonas putida KT2440 via secretomic analysis

Understanding of Bacterial Lignin Extracellular Degradation Mechanisms by Pseudomonas putida KT2440 via Secretomic Analysis

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