Insight into the oligomeric structure of PORA from A. thaliana.
Light-dependent protochlorophyllide oxidoreductase (POR, E.C. 1.3.1.33) is a plant enzyme that directly needs light to conduct a biochemical reaction. In the present paper we confirmed that POR forms large oligomers in solution before binding of substrates. We carried out the research using different techniques: cross-linking, native gel electrophoresis and FRET measurements. Mass spectrometry analysis of the cross-link products provided the first structural data about the organisation of the oligomer of POR. The results indicated that the catalytic motifs of the adjacent subunits become close to each other upon binding of substrates. Moreover, we identified two mutations of POR that disturbed its oligomerisation properties: Δ85-88 and Δ240-270. Additionally, a complete loss of the catalytic activity was observed for the following mutations: Δ189-194, Δ240-270, Δ318-331 and Δ392-393.
Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: michal.gabruk@uj.edu.pl.
Michal
M
Gabruk
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
Zuzanna
Z
Nowakowska
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Kraków, Poland.
Bozena
B
Skupien-Rabian
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Kraków, Poland.
Sylwia
S
Kędracka-Krok
Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
Beata
B
Mysliwa-Kurdziel
Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: jerzy.kruk@uj.edu.pl.
Jerzy
J
Kruk
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003432:Cross-Linking Reagents; D031541:Fluorescence Resonance Energy Transfer; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase
Cross-linking; Oligomerisation; Protochlorophyllide oxidoreductase