Characterization of a plant S-adenosylmethionine synthetase from Acacia koa
Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.
| Veröffentlicht in: | Plant physiology and biochemistry : PPB. - 1991. - 210(2024) vom: 09. Mai, Seite 108618 |
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| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2024
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| Zugriff auf das übergeordnete Werk: | Plant physiology and biochemistry : PPB |
| Schlagworte: | Journal Article Feedback inhibition Heterologous expression Hybrid enzyme Non-aqueous activity assay Recombinant enzyme Methionine Adenosyltransferase EC 2.5.1.6 Plant Proteins S-Adenosylmethionine |
| Zusammenfassung: | Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved. The Acacia koa S-adenosylmethionine (SAM) synthetase was identified from transcriptome data and cloned into the T7-expression vector pEt14b. Assays indicate a thermoalkaliphic enzyme which tolerates conditions up to pH 10.5, 55 °C and 3 M KCl. In vitro examples of plant SAM-synthetase activity are scarce, however this study provides supporting evidence that these extremophilic properties may actually be typical for this plant enzyme. Enzyme kinetic constants (Km = 1.44 mM, Kcat = 1.29 s-1, Vmax 170 μM. min-1) are comparable to nonplant SAM-synthetases except that substrate inhibition was not apparent at 10 mM ATP/L-methionine. Methods were explored in this study to reduce feedback inhibition, which is known to limit SAM-synthetase activity in vitro. Four single-point mutation variants of the Acacia koa SAM-synthetase were produced, each with varying degrees of reduced reaction rate, greater sensitivity to product inhibition and loss of thermophilic properties. Although an enhanced mutant was not produced, this study describes the first mutagenesis of a plant SAM-synthetase. Overcoming feedback inhibition was accomplished by the addition of organic solvent to enzyme assays. Acetonitrile, methanol or dimethylformamide, when included as 25% of the assay volume, improved total SAM production by 30-65% |
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| Beschreibung: | Date Completed 09.05.2024 Date Revised 09.05.2024 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1873-2690 |
| DOI: | 10.1016/j.plaphy.2024.108618 |