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Structural Requirements
for Enzymatic Activity in a Single Catalytic Domain of a Dinoflagellate
Luciferase
Liu, Liyun1
and Hastings, J. Woodland1
Harvard University1
Abstract-
Luciferase from Gonyaulax polyedra is highly unusual; there are
three catalytic sites in its single chain (~137kDa), which are located
in the central regions (aa 127-281; >95% identical) of each of three
contiguous homologous 377-aa domains. When separately cloned and expressed
in E. coli as fusion proteins the full length domains are enzymatically
active. The second domain has been used to examine the structural requirements
for activity and pH regulation. Up to at least 66 N-terminal aa can
be removed without a full loss of activity, and within this region four
intramolecularly conserved histidines are implicated in the loss of
activity at pH 8 in the full length peptide. Their replacement singly
or in groups by alanine by site directed mutagenesis resulted in peptides
which retained good activity at pH 6.3 (the pH optimum) but greatly
increased activities at pH 8. The region in which the four histidines
is located (aa 30-82) is more highly conserved (64% identical) than
sequences before (1-29, 28%) and after (83-114, 31%), indicative of
its importance. The peptide from aa 115 to the C-terminus (377) is inactive.
At the C-terminus 20 aa can be removed without loss of activity or change
in pH-activity profile, but peptides with 40 aa removed are inactive.
Here again there is a difference in conservation: the first 20 C-terminal
aa share only 30% identity with the other two domains, whereas the next
20 share 60%. The presence of another island of high conservation (aa
290-328;~90%) suggests that the active site may involve more than the
central region (aa 127-281). In that region there is also an unexplained
nucleotide conservation: synonymous substitutions are far less common
than in the flanking regions.
Keywords: dinoflagellate,
luciferase, pH/activity profile, conserved residues
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