UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli.
Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.
Warnecke
D C
DC
Institut für Allgemeine Botanik, University of Hamburg, Germany.
Baltrusch
M
M
Buck
F
F
Wolter
F P
FP
Heinz
E
E
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D018554:Avena / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins; D013261:Sterols / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D018744:DNA, Plant; D011993:Recombinant Fusion Proteins; D013261:Sterols; D005964:Glucosyltransferases; C085360:sterol 3-beta-glucosyltransferase