PLANT PHYSIOLOGY , Vol 110, Issue 4 1197-1205, Copyright © 1996 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Characterization of Soybean Choline Kinase cDNAs and Their Expression in Yeast and Escherichia coli
D. E. Monks, J. H. Goode and R. E. Dewey
Department of Crop Science, North Carolina State University, Raleigh, North Carolina 27695-7620
An expressed sequence tag from Arabidopsis that displayed sequence homology
to mammalian and yeast choline kinases was used to isolate choline
kinase-like cDNAs from soybean (Glycine max L.). Two distinct cDNAs,
designated GmCK1 and GmCK2, were recovered that possessed full-length
reading frames, each sharing approximately 32% identity at the predicted
amino acid level with the rat choline kinase. A third unique choline
kinase-like cDNA, GmCK3, was also identified but was not full length.
Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and
GmCK2 in both yeast and Escherichia coli demonstrated that each encodes
choline kinase activity. In addition to choline, other potential substrates
for the choline kinase enzyme include ethanolamine, monomethylethanolamine
(MME), and dimethylethanolamine (DME). Both soybean choline kinase isoforms
demonstrated negligible ethanolamine kinase activity. Competitive
inhibition assays, however, revealed very distinct differences in their
responses to DME and MME. DME effectively inhibited only the GmCK2-encoded
choline kinase activity. Although MME failed to effectively inhibit either
reaction, an unexpected enhancement of choline kinase activity was observed
specifically with the GmCK1-encoded enzyme. These results show that choline
kinase is encoded by a small, multigene family in soybean comprising two or
more distinct isoforms that exhibit both similarities and differences with
regard to substrate specificity.
