THESIS
2013
xv leaves, 75 pages : illustrations (some color) ; 30 cm
Abstract
In the first part of this dissertation, the structural studies of Cba3 were described.
Cellulose is the main structural component of the plant cell wall, the most abundant
polysaccharide on Earth, and an important renewable resource. It consists of D-glucose
residues linked by β-1,4-glycosidic bonds to form linear polymeric chains of over
10,000 glucose residues. Enzymatic conversion of cellulose to glucose is a complicated
process and involves cooperative action of three enzymes: endoglucanas, exoglucanase,
and cellobiase. Cba3, characterized from Cellulomonas biazotea in 2012, was a
cellobiase catalyzing the breakdown reaction of one cellobiose into two glucoses.
Previous sequence alignment studies reveal that Cba3 belongs to GH1 (Glycoside
hydrolases 1) family and it is the...[
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In the first part of this dissertation, the structural studies of Cba3 were described.
Cellulose is the main structural component of the plant cell wall, the most abundant
polysaccharide on Earth, and an important renewable resource. It consists of D-glucose
residues linked by β-1,4-glycosidic bonds to form linear polymeric chains of over
10,000 glucose residues. Enzymatic conversion of cellulose to glucose is a complicated
process and involves cooperative action of three enzymes: endoglucanas, exoglucanase,
and cellobiase. Cba3, characterized from Cellulomonas biazotea in 2012, was a
cellobiase catalyzing the breakdown reaction of one cellobiose into two glucoses.
Previous sequence alignment studies reveal that Cba3 belongs to GH1 (Glycoside
hydrolases 1) family and it is the first discovered GH1 β-glucosidase of C. biazotea. As
the studies looking for high efficient cellobiase for industrial application are extensively
increased, the structural studies of Cba3 would provide insights into the engineering of
enzymes for enhanced protein stability and increased activity. On the other hand, as
Cba3 presents different enzymatic activities towards cellobiose and lactose, the
structural studies of Cba3 with its substrates would be greatly helpful for the elucidation
of mechanism of substrate specificity.
We expressed, purified and crystallized Cba3. We also resolved the three
dimensional structure of Cba3. By the analysis of its structure, we found that Cba3
adopts classic (β/α)
8 barrel fold. According to the experiment result, we make some
primary discussion about its biological function.
The second part of this dissertation describes the biochemical and structural studies
of human Orc6 involved in origins recognition and binding during DNA replication
initiation process. DNA replication initiation in eukaryotes requires a series of steps
raging from origin recognition by ORC (origin recognition complex) to the activation of
DNA helicase. The whole process is highly regulated to ensure that DNA is replicated a
single round during each cell cycle which is crucial for the maintenance of the genome
integrity. Through increasing studies on eukaryotes from yeast to human, it is indicated
that the fundamental mechanism of DNA replication is well conserved. Orc6 is the least
conserved of all ORC subunits. Sequence alignment between budding yeast and
metazoan do not show statistically significant homology. In budding yeast, Orc6 is
significantly larger than in metazoan species. Various studies show that Orc6 plays
different functions in different species. In budding yeast Orc6 is dispensible for DNA
binding in vitro. Further studies even report that yeast Orc6 interacts with Cdt1 and is
involved in the loading of MCM helicase. In Xenopus and humans, Orc6 does not seem
to be tightly associated with other core ORC subunits. Nevertheless, Orc6 is an integral
part of Drosophila ORC and is essential for both DNA binding and replication both in
vitro and in vivo. Moreover, Orc6 is reported to be implicated in cytokinesis and
chromosome segregation in both Drosophila and human cells. Since Orc6 plays so
many roles, the structure determination of Orc6 would be very helpful to study its
functions. In 2011, the structure of the middle region of human Orc6 (94-187) has been
determined. The researches showed that Orc6 middle region has an overall fold similar
to the corresponding helical domain of transcription factor TFIIB. Then they proposed a
model about how Orc6 interact with DNA. They further suggested that Orc6 directly
binds with DNA in metazoans. As the full length of human Orc6 and other fragments were not easily crystallized and the complex structure of Orc6 middle region with DNA
is still a model based on bioinformatics, therefore, more researches are required to
understand its biological functions. We mainly focused on structural studies of Orc6
middle region with DNA and other fragments by nuclear magnetic resonance
spectroscopy (NMR) method.
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