Plant Molecular Biology 35: 735–748, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Lysine-ketoglutarate reductase and saccharopine dehydrogenase from
Arabidopsis thaliana: nucleotide sequence and characterization
, Raymond McDevitt and S. Carl Falco
Agricultural Products, E.I. DuPont de Nemours & Co., Wilmington, DE 19880-0402, USA (
Received 4 April 1997; accepted in revised form 7 July 1997
Key words: Arabidopsis thaliana, heterologous expression, lysine catabolism, lysine-ketoglutarate reductase sac-
We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 18.104.22.168) and saccharopine dehydrogenase
(SDH, ED 22.214.171.124) from an Arabidopsis thaliana genomic DNA library based on the homology between the
yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed
sequence tags. A correspondingcDNA was isolated from total Arabidopsis RNA using RT-PCR and 5
DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH
(lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming).
Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs
showed 25% and 37% sequence identity, respectively. No intracellular targeting sequence was found at the N-
terminal or C-terminal of the protein. The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is
present in Arabidopsis in a single copy. 5
sequence analysis revealed several conserved promoter sequence motifs,
but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box. The 3
-ﬂanking region does
not contain a polyadenylation signal resembling the consensus sequence AATAAA. The plant SDH was expressed
in Escherichia coli and exhibited similar biochemical characteristics to those reported for the puriﬁed enzyme from
maize. This is the ﬁrst report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.
Abbreviations: AK, aspartate kinase; CTP, chloroplast transit peptide; DHDPS, dihydrodipicolinate synthase;
LKR, lysine-ketoglutarate reductase; SDH, saccharopine dehydrogenase.
Lysine is synthesizedin higherplants andin many bac-
terial species from aspartate [6, 8]. Its rate of synthesis
in plants is regulated mainly by feedback inhibition
of aspartate kinase (AK) and dihydrodipicolinate syn-
thase (DHPS) . These enzymes therefore play an
important role in determining the level of free lysine.
Control of the biosynthetic pathwayto lysine is of spe-
cial interest, since lysine levels are low in the seeds of
The nucleotide sequence data reported will appear in the Gen-
Bank, EMBL and DDBJ Nucleotide Sequence Databases under the
accession numbers U95758 (A. thaliana (Landsberg erecta) LKR-
SDH gene) and U95759 (A. thaliana (Columbia) LKR-SDH gene).
importantcrop plants, such as corn, thereby decreasing
its nutritional quality .
Expression of feedback insensitive bacterial DHPS
has been shown to result in elevated levels of free lys-
ine in canola, soybean, and maize seeds [9, Falco et al.,
unpublished results]. In each case the increased level
of free lysine is accompanied by accumulation of the
lysine breakdown products saccharopine or
adipic acid. Lysine-ketoglutarate reductase (LKR, EC
126.96.36.199) and saccharopine dehydrogenase (SDH, EC
188.8.131.52) catalyze the ﬁrst and second step, respect-
ively, in thebreakdownpathway of lysine that produces
these intermediates in seeds (Figure 1) . LKR con-
denses lysine and
-ketoglutarate into saccharopine