| 1. |
- Acebes, S., et al.
(författare)
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Mapping the Long-Range Electron Transfer Route in Ligninolytic Peroxidases
- 2017
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 121:16, s. 3946-3954
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Tidskriftsartikel (refereegranskat)abstract
- Combining a computational analysis with site-directed mutagenesis, we have studied the long-range electron transfer pathway in versatile and lignin peroxidases, two enzymes of biotechnological interest that play a key role for fungal degradation of the bulky lignin molecule in plant biomass. The in silico study established two possible electron transfer routes starting at the surface tryptophan residue previously identified as responsible for oxidation of the bulky lignin polymer. Moreover, in both enzymes, a second buried tryptophan residue appears as a top electron transfer carrier, indicating the prevalence of one pathway. Site-directed mutagenesis of versatile peroxidase (from Pleurotus eryngii) allowed us to corroborate the computational analysis and the role played by the buried tryptophan (Trp244) and a neighbor phenylalanine residue (Phe198), together with the surface tryptophan, in the electron transfer. These three aromatic residues are highly conserved in all the sequences analyzed (up to a total of 169). The importance of the surface (Trp171) and buried (Trp251) tryptophan residues in lignin peroxidase has been also confirmed by directed mutagenesis of the Phanerochaete chrysosporium enzyme. Overall, the combined procedure identifies analogous electron transfer pathways in the long-range oxidation mechanism for both ligninolytic peroxidases, constituting a good example of how computational analysis avoids making extensive trial-error mutagenic experiments.
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| 2. |
- Lambrughi, Matteo, et al.
(författare)
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DEAMINATION REACTIONS AS PART OF THE METABOLIC PATHWAY FOR THE PRODUCTION OF ADIPIC ACID
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Deamination of lysine, β-lysine or 2-aminoadipic acid are enzymatic reactions that have great biotechnological interest as part of the metabolic pathways for the production of adipic acid. The enzymatic activity necessary to catalyze the mentioned deamination reactions is defined as ammonia lyase (EC 4.3.1.-) and cleaves the bond between the amine group and adjacent carbon. Whereas ammonia lyases able to act on the target substrates lysine, β-lysine or 2-aminoadipic acid have not been identified so far, ammonia lyases able to act on other amino acids have long been known. We selected 3-methylaspartate-ammonia lyase (MAL, EC 4.3.1.2) as enzyme that potentially could be made active on the target substrates. Three MAL were recombinantly expressed and purified and the activity tested towards the substrates, but no activity was observed. Different MAL single mutant variants potentially able to catalyze the desired reactions were designed using a computational approach, but unfortunately, no activity towards the target substrates was detected. In order to better understand the substrate scope and catalytic mechanism of MAL, different compunds with similar structure to the natural substrate were tried as substrates. MAL showed activity towards aspartic acid, but no towards the other substrates indicating the narrow specificity of the enzyme. Inhibition studies showed that β-lysine was a competitive inhibitor suggesting that the amino group of the substrate need to be in the β-carbon for the binding. 2-aminoadipic acid was shown to be a non-competitive inhibitor. Finally, docking experiments were carried out to understand if the target substrates fit in the catalytic pocket. The study provides a deeper knowledge of the substrate scope and inhibitors of MAL and analyze if and how the target substrates could be deaminated by MAL. Moreover, the study establishes reliable methods for the detection of deamination activity of lysine, β-lysine and 2-aminoadipic acid.
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| 3. |
- Saez Jimenez, Veronica, 1985, et al.
(författare)
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Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude
- 2022
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:8, s. 2779-2790
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Tidskriftsartikel (refereegranskat)abstract
- Pathway engineering is commonly employed to improve the production of various metabolites but may incur in bottlenecks due to the low catalytic activity of a particular reaction step. The reduction of 2-oxoadipate to (R)-2-hydroxyadipate is a key reaction in metabolic pathways that exploit 2-oxoadipate conversion via α-reduction to produce adipic acid, an industrially important platform chemical. Here, we engineered (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) with the aim of improving 2-oxoadipate reduction. Using a combination of computational analysis, saturation mutagenesis, and random mutagenesis, three mutant variants with a 100-fold higher catalytic efficiency were obtained. As revealed by rational analysis of the mutations found in the variants, this improvement could be ascribed to a general synergistic effect where mutation A206V played a key role since it boosted the enzyme's activity by 4.8-fold. The Hgdh variants with increased activity toward 2-oxoadipate generated within this study pave the way for the bio-based production of adipic acid.
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| 4. |
- Saez Jimenez, Veronica, 1985, et al.
(författare)
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Engineering ammonia-lyases for lysine transformation: first steps to green production of adipic acid
- 2017
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Adipic acid is one of the most important dicarboxylic acid for commercial purposes, mainly used as building block for nylon polymers. The current chemical production process has serious consequences for the environment. Therefore, the implementation of a by a bio-based process using renewable feedstocks would be highly beneficial for the society and the environment. The construction of a microbial metabolic pathway to produce adipic acid using L-lysine as precursor is a potential alternative. The first step of the proposed pathway converts L-lysine into 6-aminohex-2-enoic acid (6-AHEA) via removal of the α-amino group. Chemical methods for this deamination reaction are known; however, no enzymes able to carry out this reaction on L-lysine have been disclosed yet. The main goal of the current research is the generation a novel enzyme activity for the conversion of L-lysine to 6-AHEA. The enzymatic activity necessary to catalyze the required deamination is defined as ammonia lyase and results in the removal of the α-amino group. Histidine ammonia-lyase (HAL) and 3-methylaspartate-ammonia-lyase (MAL), enzymes acting on histidine and 3-methylaspartate, respectively, were selected to be engineered to catalyze the deamination of lysine. HAL from Pseudomonas putida and MAL from Clostridium tetanomorphum and Carboxydothermus hydrogenoformans were expressed in E.coli and purified. The capability of the enzymes to deaminate lysine was tested. No deamination activity was observed, while the inhibitory effect of L-lysine on HAL activity was shown. Computational structural biology methodologies were applied on MAL and combined with protein engineering techniques in order to design mutant enzyme variants potentially active on L-lysine. In-silico saturation mutagenesis tools were used to model all the possible mutations in the active site or its surroundings that are expected to increase affinity for the L-lysine substrate. Following the results obtained, the residues C361, M389 and L384 were mutagenized. The mutant variants were produced and purified, and the activity on L-lysine tested by monitoring the production of 6-AHEA and the release of ammonia.
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| 5. |
- Saez Jimenez, Veronica, 1985, et al.
(författare)
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Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin
- 2016
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility. Results: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two-and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k(3app) values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have similar to 20-30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used. Conclusions: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.
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| 6. |
- Saez Jimenez, Veronica, 1985, et al.
(författare)
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Structure-function investigation of 3-methylaspartate ammonia lyase reveals substrate molecular determinants for the deamination reaction
- 2020
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 15:5
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Tidskriftsartikel (refereegranskat)abstract
- The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.
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| 7. |
- Skoog, Emma, 1983, et al.
(författare)
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Biobased adipic acid – The challenge of developing the production host
- 2018
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 36:8, s. 2248-2263
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Forskningsöversikt (refereegranskat)abstract
- Adipic acid is a platform chemical, and is the most important commercial dicarboxylic acid. It has been targeted for biochemical conversion as an alternative to present chemical production routes. From the perspective of bioeconomy, several kinds of raw material are of interest including the sugar platform (derived from starch, cellulose or hemicellulose), the lignin platform (aromatics) and the fatty acid platform (lipid derived). Two main biochemical-based production schemes may be employed: (i) direct fermentation to adipic acid, or (ii) fermentation to muconic or glucaric acid, followed by chemical hydrogenation (indirect fermentation). This review presents a comprehensive description of the metabolic pathways that could be constructed and analyzes their respective theoretical yields and metabolic constraints. The experimental yields and titers obtained so far are low, with the exception of processes based on palm oil and glycerol, which have been reported to yield up to 50 g and 68 g adipic acid/L, respectively. The challenges that remain to be addressed in order to achieve industrially relevant production levels include solving redox constraints, and identifying and/or engineering enzymes for parts of the metabolic pathways that have yet to be metabolically demonstrated. This review provides new insights into ways in which metabolic pathways can be constructed to achieve efficient adipic acid production. The production host provides the chassis to be engineered via an appropriate metabolic pathway, and should also have properties suitable for the industrial production of adipic acid. An acidic process pH is attractive to reduce the cost of downstream processing. The production host should exhibit high tolerance to complex raw material streams and high adipic acid concentrations at acidic pH.
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