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  1. Article ; Online: Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

    Vargas, Jhon A / Sculaccio, Susana A / Pinto, Andressa P A / Pereira, Humberto D'Muniz / Mendes, Luis F S / Flores, Jhoao F / Cobos, Marianela / Castro, Juan C / Garratt, Richard C / Leonardo, Diego A

    Journal of experimental botany

    2024  Volume 75, Issue 9, Page(s) 2754–2771

    Abstract: l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its ... ...

    Abstract l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.
    MeSH term(s) Ascorbic Acid/metabolism ; Ascorbic Acid/biosynthesis ; Fruit/metabolism ; Plant Proteins/metabolism ; Plant Proteins/genetics ; Plant Proteins/chemistry ; Myrtaceae/metabolism ; Myrtaceae/genetics ; Galactose Dehydrogenases/metabolism ; Galactose Dehydrogenases/genetics ; Oxidoreductases Acting on CH-CH Group Donors/metabolism ; Oxidoreductases Acting on CH-CH Group Donors/genetics
    Chemical Substances Ascorbic Acid (PQ6CK8PD0R) ; Plant Proteins ; galactonolactone dehydrogenase (EC 1.3.2.3) ; galactose dehydrogenase (EC 1.1.1.48) ; Galactose Dehydrogenases (EC 1.1.1.-) ; Oxidoreductases Acting on CH-CH Group Donors (EC 1.3.-)
    Language English
    Publishing date 2024-05-02
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2976-2
    ISSN 1460-2431 ; 0022-0957
    ISSN (online) 1460-2431
    ISSN 0022-0957
    DOI 10.1093/jxb/erae016
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Bonn / Germany

    Z 55/29: Show issues

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  2. Article ; Online: Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange.

    Blázquez-Sánchez, Paula / Vargas, Jhon A / Furtado, Adriano A / Griñen, Aransa / Leonardo, Diego A / Sculaccio, Susana A / Pereira, Humberto D'Muniz / Sonnendecker, Christian / Zimmermann, Wolfgang / Díez, Beatriz / Garratt, Richard C / Ramírez-Sarmiento, César A

    Protein science : a publication of the Protein Society

    2023  Volume 32, Issue 9, Page(s) e4757

    Abstract: Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic ... ...

    Abstract Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.
    MeSH term(s) Polyethylene Terephthalates/chemistry ; Polyethylene Terephthalates/metabolism ; Antarctic Regions ; Hydrolases/chemistry ; Hydrolysis ; Protein Engineering ; Plastics
    Chemical Substances Polyethylene Terephthalates ; Hydrolases (EC 3.-) ; Plastics
    Language English
    Publishing date 2023-08-13
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1106283-6
    ISSN 1469-896X ; 0961-8368
    ISSN (online) 1469-896X
    ISSN 0961-8368
    DOI 10.1002/pro.4757
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 3407: Show issues Location:
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    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
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  3. Article ; Online: Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

    Vargas, Jhon A / Leonardo, Diego A / D'Muniz Pereira, Humberto / Lopes, Adriana R / Rodriguez, Hicler N / Cobos, Marianela / Marapara, Jorge L / Castro, Juan C / Garratt, Richard C

    Plant & cell physiology

    2022  Volume 63, Issue 8, Page(s) 1140–1155

    Abstract: In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component ... ...

    Abstract In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.
    MeSH term(s) Ascorbic Acid/metabolism ; Galactose/metabolism ; Galactose Dehydrogenases/metabolism ; Mannose/metabolism ; NAD
    Chemical Substances NAD (0U46U6E8UK) ; Galactose Dehydrogenases (EC 1.1.1.-) ; Mannose (PHA4727WTP) ; Ascorbic Acid (PQ6CK8PD0R) ; Galactose (X2RN3Q8DNE)
    Language English
    Publishing date 2022-07-13
    Publishing country Japan
    Document type Journal Article
    ZDB-ID 208907-5
    ISSN 1471-9053 ; 0032-0781
    ISSN (online) 1471-9053
    ISSN 0032-0781
    DOI 10.1093/pcp/pcac090
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Bonn / Germany

    Z 79/710: Show issues

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  4. Article: Genomic analysis and biochemical profiling of an unaxenic strain of

    Cobos, Marianela / Condori, Ruth C / Grandez, Miguel A / Estela, Segundo L / Del Aguila, Marjorie T / Castro, Carlos G / Rodríguez, Hicler N / Vargas, Jhon A / Tresierra, Alvaro B / Barriga, Luis A / Marapara, Jorge L / Adrianzén, Pedro M / Ruiz, Roger / Castro, Juan C

    Frontiers in genetics

    2022  Volume 13, Page(s) 973324

    Abstract: Cyanobacteria are diverse photosynthetic microorganisms able to produce a myriad of bioactive chemicals. To make possible the rational exploitation of these microorganisms, it is fundamental to know their metabolic capabilities and to have genomic ... ...

    Abstract Cyanobacteria are diverse photosynthetic microorganisms able to produce a myriad of bioactive chemicals. To make possible the rational exploitation of these microorganisms, it is fundamental to know their metabolic capabilities and to have genomic resources. In this context, the main objective of this research was to determine the genome features and the biochemical profile of
    Language English
    Publishing date 2022-11-09
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2606823-0
    ISSN 1664-8021
    ISSN 1664-8021
    DOI 10.3389/fgene.2022.973324
    Database MEDical Literature Analysis and Retrieval System OnLINE

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