DOCTORAL THESIS

Biochemical Studies on Ferredoxin-NADP+Oxidoreductase (FNR) and Thermostable
Glucosidase in Cyanobacteria.
Graduate School of
Natural Science and Technology,Kanazawa UniversityMajor Subject: BotanySpecial Subject (Course): Plant Biochemistry

School registration No. (0323032326)
Author name: Fatthy Mohamed Sayed Morsy
Chief advisor: Associate Prof. Dr. Toshio Sakamoto

Abstract
Ferredoxin-NADP+ oxidoreductase (FNR) catalyzing the terminal step of thelinear photosynthetic electron transport was purified to homogeneity from thecyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR ofSpirulina consisted of three domains (CpcD-like domain, FAD-binding domain andNADP+-binding domain) with a molecular mass of 46 kDa and was localized in either
phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa
was solubilized by NaCl and the solublized FNR had an apparent molecular mass of90 kDa. Degradation of Spirulina 46 kDa FNR in vitro produced the intermediate size
FNRs with molecular masses of 37 kDa, 36 kDa, 35 kDa and 34 kDa and thesecleavages of FNR protein specifically took place in the hinge region, which is theinterspace region between the CpcD-like domain and the catalytic domains according
to the N-terminal amino-acid sequences of these intermediate size FNRs. Theseresults suggest that the cyanobacterial two-domains FNR is a proteolytic product of
the three domains cyanobacterial FNR. FNR of Cyanidium consisted of two domains(FAD-binding domain and NADP+-binding domain) with a molecular mass of 33 kDa.
In Cyanidium, FNR was found on thylakoid membranes but there was no FNR on
phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized byNaCl, suggesting the enzyme is tightly bound in the membrane. All forms of FNR
under investigation in this study were biochemically similar and showed a highly
active quinone dependent cytochrome C reduction activity indicating that they have aquinone reductase activity as well as ferredoxin-NADP+ oxidation reduction activity.
Although both cyanobacteria and red algae are photoautotrophic organisms bearingiphycobilisomes as the light harvesting complexes, FNR localization andcharacteristics of binding to the membrane were different. These results suggest thatFNR binding to phycobilisomes is not characteristic for all phycobilisome retaining
oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly
originated from a cyanobacterium ancestor with FNR lacking the CpcD-like domain.
The cyanobacterium Nostoc commune is adapted to the terrestrial environment
and forms a visible size of colony in which the cells are surrounded by extracellularpolysacharides (EPS). The EPS play a crucial role for extreme desiccation toleranceof this organism while the EPS-depleted cells are sensitive to desiccation. When thenatural colonies immersed in water at room temperature, breakdown of colonies wasobserved within 2 days and fully vital N. commune cells with photosynthetic O2evolution and respiratory O2 consumption were released to the water. In this study, a
new method for massive isolation of the external polysaccharides (EPS) from cells of
the terrestrial cyanobacterium Nostoc commune colonies was developed using highconcentration phosphate buffer. The EPS-depleted cells of N. commune represented
4.7 % of the total fresh mass of N. commune colonies and around 95 % of the total
fresh mass of N. commune was in the extracted water hydrated external
polysaccharides. Around 96 % of the total protein and almost no EPS were recoveredin the EPS-depleted cells. The activities to hydrolyze the glycoside bonds using
artificial nitrophenyl-linked sugars as substrates were examined. A thermostable beta
D-glucosidase activity was detected in the water soluble fraction of the EPS and theenzyme was resistant to boiling for 20 minuets. The beta D-glucosidase was purified
to homogeneity. The molecular mass of the purified protein was 20 kDa and its
N-terminal amino-acid sequence was identical with the cyanobacterial secreted andsurface fasciclin protein. The beta D-glucosidase activity of the purified protein had aii

neutral optimum pH value of 7 and showed a lower affinity towards other glycosidebonds. The gene encoding the fasciclin protein was amplified and sequenced from N.
commune strain KU002, N. punctiforme strain M15 and N. commune (D-type), andthe deduced amino-acids sequence showed a high similarity with the protein of othercyanobacterial species. The calculated molecular mass of the deduced protein in N.
commune starin KU002, N. punctiforme strain M15 and N. commune (D-type) were
13.55 kDa, 13.58 kDa and 13.6 kDa respectively. The slow electrophoretic migrationof the native protein (20 kDa) might indicate a possible posttranslational modificationin the native protein. The glucosidase activity of the fasciclin protein in N. commune
might play a role in partial hydrolysis of the EPS surrounding the N. commune cells
possibly for growth of the colony and cell expansion.

Acknowledgements
All gratitude is due to "GOD", the almighty,
who guided and aided me bring forth to light thiswork.
I would like to thank Prof. Dr. Keishiro Wada and Associate Prof.
Dr. Toshio Sakamoto for their valuable scientific advices. Sincere thanks
for Associate Prof. Dr. Toshio Sakamoto for continuous supervising of
this work for the Ph.D degree of science. I appreciate theirencouragement. I am grateful for the camaraderie in the lab with othermembers, Takayuki Yoshida, Tatsuki Fujiwara and Arima Hiromi, inparticular for their kind help and valuable discussions. Collaborationiii

with undergraduate and graduate students in the lab has been achallenge and an inspiration since we always seem to learn from eachother. My Deep thanks for Associate Prof. Dr. Hoshina Satoshi and Dr.
Masato Nakajima for their valuable discussion. Sincere thanks forMisses Sanada Yukika for kind helps. My sincere thanks for Prof. Dr.
Fukumori Yoshihiro and his lab members, Taoka Azuma and Nakagawa
Tarou, in particular for their kind help in using many tools in theirlaboratory. My deep thanks for Prof Dr. Endoh Hiroshi and prof. Dr.
Sakurai Syo for their continuous encouragements and advices. Sincere
thanks for Associate Prof. Dr. Kanemori Masaaki and Associate Prof. Dr.
Yamaguchi Masaaki for their good dealing and encouragements. My
deep thanks for all staff and students in the Department of biology for
their good dealing and nice support in scientific and social activities. This
researchwasmadepossible

throughthesupportoftheMONBOKAGAKSHO and the EGYPTIAN GOVERNMENT MISSION
DEPARTMENT.

Fatthy M. S. Morsyiv