Plant type II NAD(P)H dehydrogenases : Structure, regulation and evolution of NDB proteins
Author
Summary, in English
Plant NDB-type proteins are NDH-2 enzymes located at the external mitochondrial inner membrane. It was earlier found that NDB1 oxidise cytosolic NADPH, and NDB2 oxidise cytosolic NADH. In this study, the regulatory mechanisms of Arabidopsis thaliana and Solanum tuberosum NDB1 by cytosolic Ca2+ and pH were studied and compared to NDB2, using purified mitochondria and E. coli-produced proteins in a membrane-bound and a purified soluble state. Membrane bound NDB1 and NDB2 oxidised NADPH and NADH, respectively. Soluble forms of NDB1 oxidise both NADH and NADPH, with higher NADPH activity. Soluble forms of NDB2 oxidised only NADH like the membrane-bound enzyme. In solution, the active StNDB1 resided as oligomers of dimeric units, mainly hexamers, and recombinant AtNDB2 was highly oligomeric. Within a physiological pH range, an acidic pH was found to lower the Ca2+ demand for activation of the mitochondrial and E. coli-produced NADPH oxidation via NDB1, as compared to a more alkaline pH. Depending on pH, 3-82 µM Ca2+ was needed. In contrast, the sub-µM Ca2+ demand for activation of NADH oxidation was not linked to pH. Both soluble and mitochondrial StNDB1 (NADPH oxidation) could respond quickly to increased and decreased Ca2+, whereas mitochondrial NADH oxidation responded quickly to Ca2+ increase but slowly to Ca2+ decrease. Overall, the results suggest that in vivo, the activity of NDB1 is rapidly controlled by pH-shift-associated Ca2+ spikes in the cytosol whereas NDB2 may be more continuously active.
Based on modelling of NDB1, the core catalytic parts and dimerization surface showed distinct similarities to the structures of yeast ScNDI1 and Plasmodium falciparum PfNDH-2. This analysis highlighted motifs that correlate with NAD(P)H substrate specificity, and which were followed by evolutionary analysis. Most eukaryotic species have NDB proteins that contain a non-acidic motif for NADPH binding. Angiosperms and liverworts contain NDB proteins of NDB1- and NDB2- type, i.e. they contain acidic and non-acidic motifs for NADH and NADPH binding, respectively. This indicates that plants have more flexibility for external NAD(P)H oxidation as compared to other eukaryotes. Based on the evolutionary analysis, Ca2+-dependent external NADPH oxidation appears to be an ancient process as compared to NADH oxidation, and thus possibly has a more fundamental function in cellular redox metabolism.
Department/s
Publishing year
2019-05-03
Language
English
Full text
Document type
Dissertation
Publisher
Lund University, Faculty of Science, Department of Biology
Topic
- Cell Biology
Keywords
- Arabidopsis
- Ca2+
- Electron transport
- NDH-2
- NDB
- pH
- NADH
- NADPH
- Plant mitochondria
- Potato
- Type II NAD(P)H dehydrogenase
Status
Published
Supervisor
ISBN/ISSN/Other
- ISBN: 978-91-7753-975-9
- ISBN: 978-91-7753-974-2
Defence date
29 May 2019
Defence time
09:30
Defence place
Biology lecture hall (A213), the Biology building, Sölvegatan 35, Lund
Opponent
- Tom Hamborg Nielsen (Associate Professor)