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PEDS Advance Access originally published online on July 14, 2006
Protein Engineering Design and Selection 2006 19(10):443-452; doi:10.1093/protein/gzl028
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Probing conserved amino acids in phospholipase D (Brassica oleracea var. capitata) for their importance in hydrolysis and transphosphatidylation activity

Alexandra Lerchner, Johanna Mansfeld, Konstantin Kuppe and Renate Ulbrich-Hofmann1

Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology Kurt-Mothes-Strasse 3, D-06120 Halle, Germany

1To whom correspondence should be addressed. E-mail: Ulbrich-Hofmann{at}biochemtech.uni-halle.de

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.

Keywords: Brassica oleracea var. capitata/E.coli/phospholipase D/mutagenesis/transphosphatidylation

Received February 14, 2006; revised May 19, 2006; accepted June 12, 2006.


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