Photoactive protochlorophyllide-enzyme complexes reconstituted with PORA, PORB and PORC proteins of A. thaliana : fluorescence and catalytic properties

Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development

Protochlorophyllide oxidoreductase - ligands interactions. A preliminary study.

Oksydoreduktaza protochlorofilidu (POR) przeprowadza jedną z ostatnich reakcji szlaku biosyntezy chlorofilu. Enzym ten w obecności światła katalizuje redukcję jednego z podwójnych wiązań pierścienia tetrapirolowego protochlorofilidu (Pchlidu), utleniając NADPH. W celu zbadania procesu wiązania barwnika do białka POR, opracowano model homologiczny białka PORA z Arabidopsis thialiana, a także otrzymano czyste i aktywne białko, ekspresjonowane w bakteriach Escherichia coli. Udało się odtworzyć charakterystyczny dla układów in vivo stan skompleksowania Pchlidu w postaci oligomerycznych kompleksów (Pchlid:PORA:NADPH)n bez udziału lipidów, PORB i Pchlidu b. Zaproponowano parametr określający stopień oligomeryzacji POR, którym był stosunek intensywności fluorescencji przy 660 nm do 630 nm, mierzonych przy wzbudzeniu falą o dł. 440 nm (I660/I630). W badanych warunkach, przy niedoborze barwnika względem enzymu, parametr ten był zależny liniowo od logarytmu dziesiętnego z ilorazu stężeń [Pchlid]:[POR]. Na podstawie opracowanego modelu wytypowano także reszty aminokwasowe, prawdopodobnie istotne w procesie wiązania barwnika. Otrzymano muteinę PORA-TT316/317AA, która miała niższy parametr opisujący zdolność do kompleksowania Pchlidu (I660/I630). Mutacja nie wpływała na wydajność redukcji barwnika. Na podstawie modelu i wyników eksperymentalnych zaproponowano, że reszty TT316/317, oddziałując z pierścieniem nikotynamidowym NADPH, stabilizują kieszeń wiążącą Pchlid.Protochlorophyllide oxidoreductase (POR) catalyses one of the last step of biochemical pathway of chlorophyll biosynthesis. In this light-induced reaction, one double bond of tetrapyrrole ring of protochlorophyllide (Pchlide) is reduced. During the same reaction one molecule of NADPH is oxidized. In order to analyze the process of pigment binding to POR, a homology model of PORA from Arabidopsis thaliana was obtained. Moreover, this enzyme was successfully purified from Escherichia coli expression system. It has been shown in this work, that (Pchlide:PORA:NADPH)n oligomers can be formed without lipids, Pchlide b or PORB in the reaction mixture. A new parameter – I660/I630 – which is the ratio of fluorescence intensity at 660 nm to that at 630 nm (read from the fluorescence spectrum measured for the excitation at 440nm) was proposed to describe the level of POR oligomeryzation. This parameter was linearly dependent on decimal logarithm of the [Pchlid]:[POR] ratio, for [Pchlid]:[POR] ratio < 1. Some residues of POR enzymes were suggested to be involved in Pchlide binding. The mutated protein PORA TT316/317AA was obtained. In this mutant, the pigment binding was weaker then in the wild type, but the mutation did not affect its catalytic properties. According to the homology model and the experimental data it was proposed that TT316/317 residues interact with nicotynamidic ring of NADPH and this interaction stabilizes the pigment-binding pocket

Chlorophyll biosynthesis: two mechanisms of protochlorophyllide reduction

W pracy podsumowano obecny stan wiedzy na temat dwu istniejących w przyrodzie mechanizmów reakcji redukcji protochlorofilidu do chlorofilidu. Redukcja protochlorofilidu jest jedną z ostatnich reakcji szlaku biosyntezy chlorofili i bakteriochlorofili - podstawowych barwników fotosyntetycznych. Reakcja ta może zachodzić w sposób niezależny od światła, katalizowany przez niezależną od światła reduktazę protochlorofilidu (DPOR) lub w procesie indukowanym światłem i katalizowanym przez zależną od światła oksydoreduktazę protochlorofilidu (LPOR). Mimo iż katalizują tę samą reakcję, enzymy LPOR i DPOR nie są spokrewnione, kodowane są przez różne geny, posiadają inną budowę cząsteczki oraz charakteryzuje je inny mechanizm katalizowanej reakcji.In the present paper, the current state of knowledge about two existing in nature mechanisms for the reduction of protochlorophyllide to chlorophyllide is presented. This reaction, which is a penultimate step of chlorophyll biosynthesis, can occur by either light-dependent or light-independent mechanisms, catalysed by light-dependent protochlorophyllide oxidoreductase (LPOR) or light-independent protochlorophyllide oxidoreductase (DPOR), respectively. LPOR and DPOR are completely different in their genes, protein structure and catalytic mechanism

Arabidopsis thaliana accessions : a tool for biochemical and phylogentical studies

Arabidopsis thaliana since a few decades is used as a model for biological and plant genetic research. Natural variation of this species is related to its geographical range which covers different climate zones and habitats. The ability to occupy such a wide area by Arabidopsis is possible due to its stress tolerance and adaptability. Arabidopsis accessions exhibit phenotypic and genotypic variation, which is a result of adaptation to local environmental conditions. During development, plants are subjected to various stress factors. Plants show a spectrum of reactions, processes and phenomena that determine their survival in these adverse conditions. The response of plants to stress involves signal detection and transmission. These reactions are different and depend on the stressor, its intensity, plant species and life strategy. It is assumed that the populations of the same species from different geographical regions acclimated to the stress conditions develop a set of alleles, which allow them to grow and reproduce. Therefore, the study of natural variation in response to abiotic stress among Arabidopsis thaliana accessions allows to find key genes or alleles, and thus the mechanisms by which plants cope with adverse physical and chemical conditions. This paper presents an overview of recent findings, tools and research directions used in the study of natural variation in Arabidopsis thaliana accessions. Additionally, we explain why accessions can be used in the phylogenetic analyses and to study demography and migration of Arabidopsis thaliana

Protochlorophylls in Cucurbitaceae : distribution, biosynthesis and phylogeny

Kruk, Jerzy, Gabruk, Michał, Kryszczak, Marian, Wąsik, Piotr (2022): Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny. Phytochemistry (113110) 197: 1-8, DOI: 10.1016/j.phytochem.2022.113110, URL: http://dx.doi.org/10.1016/j.phytochem.2022.11311