Probing conserved amino acids in phospholipase D (Brassica oleracea var. capitata) for their importance in hydrolysis and transphosphatidylation activity.
In addition to hydrolysis of glycerophospholipids, phospholipases D (PLDs) catalyze the head group exchange. The molecular basis of this transphosphatidylation potential, which strongly varies for PLDs from different sources, is unknown hitherto. Recently, the genes of two PLD isoenzymes from white cabbage have been sequenced and expressed in Escherchia coli, yielding the basis for mutational studies. In the present paper, three sequence characteristics of the isoenzyme (PLD2) that corresponds to the often used enzyme isolated from cabbage leaves have been probed for their importance in hydrolysis as well as transphosphatidylation activities: (i) the two HKD motifs, (ii) the C terminus and (iii) the eight cysteine residues. All these regions or amino acids are highly conserved in alpha-type plant PLDs. Based on multiple alignments, predictions of secondary structure and comparisons of hydrophobicity profiles, 35 enzyme variants were created and assayed. All positions tested proved to be very sensitive towards amino acid exchanges with respect to hydrolytic activity in the absence of glycerol as well as to the ratio of hydrolytic and transphosphatidylation activities in the presence of glycerol. A significant increase of total activity and transphosphatidylation activity could be obtained by the substitutions C310S and C625S.
Lerchner
Alexandra
A
Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology Kurt-Mothes-Strasse 3, Halle, Germany.
Mansfeld
Johanna
J
Kuppe
Konstantin
K
Ulbrich-Hofmann
Renate
R
D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010940:Plant Proteins; D010739:Phospholipase D