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Enzymatic Characterization of the Isocitrate Dehydrogenase with Dual Coenzyme Specificity from the Marine Bacterium Umbonibacter marinipuiceus .
- Source :
-
International journal of molecular sciences [Int J Mol Sci] 2023 Jul 13; Vol. 24 (14). Date of Electronic Publication: 2023 Jul 13. - Publication Year :
- 2023
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Abstract
- Isocitrate dehydrogenase (IDH) can be divided into NAD <superscript>+</superscript> -dependent and NADP <superscript>+</superscript> -dependent types based on the coenzyme specificity. It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from Umbonibacter Marinipuiceus (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The K <subscript>m</subscript> values for NAD <superscript>+</superscript> and NADP <superscript>+</superscript> are 1800.0 ± 64.4 μM and 1167.7 ± 113.0 μM in the presence of Mn <superscript>2+</superscript> , respectively. Meanwhile, the catalytic efficiency ( k <subscript>cat</subscript> / K <subscript>m</subscript> ) of UmIDH is only 2.3-fold greater for NADP <superscript>+</superscript> than NAD <superscript>+</superscript> . The maximal activity for UmIDH occurred at pH 8.5 (with Mn <superscript>2+</superscript> ) or pH 8.7 (with Mg <superscript>2+</superscript> ) and at 35 °C (with Mn <superscript>2+</superscript> or Mg <superscript>2+</superscript> ). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn <superscript>2+</superscript> or Mg <superscript>2+</superscript> . Moreover, three putative core coenzyme binding residues (R345, L346, and V352) of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH's coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.
Details
- Language :
- English
- ISSN :
- 1422-0067
- Volume :
- 24
- Issue :
- 14
- Database :
- MEDLINE
- Journal :
- International journal of molecular sciences
- Publication Type :
- Academic Journal
- Accession number :
- 37511187
- Full Text :
- https://doi.org/10.3390/ijms241411428