UDP-GLUCOSE-STEROL GLUCOSYLTRANSFERASE - CLONING AND FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI

Steryl glucosides are characteristic lipids of plant membranes. The bi osynthesis of these lipids is catalyzed by the membrane-bound UDP-gluc ose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Wa rnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used f or the cloning of a corresponding cDNA from oat (Avena sativa L.). Ami no acid sequences derived from the amino terminus of the purified prot ein and from peptides of a trypsin digestion were used to construct ol igonucleotide primers for polymerase chain reaction experiments. Scree ning of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-spec ific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cD NAs 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 prot ein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibite d sterol glucosyltransferase activity.