Lerchner Alexandra, Mansfeld Johanna, Kuppe Konstantin, Ulbrich-Hofmann Renate
Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology Kurt-Mothes-Strasse 3, Halle, Germany.
Protein Eng Des Sel. 2006 Oct;19(10):443-52. doi: 10.1093/protein/gzl028. Epub 2006 Jul 14.
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.
除了催化甘油磷脂水解外，磷脂酶D（PLD）还能催化头部基团交换。这种转磷脂酰化潜力的分子基础因不同来源的PLD而有很大差异，迄今为止尚不清楚。最近，已对白甘蓝中两种PLD同工酶的基因进行了测序，并在大肠杆菌中表达，为突变研究奠定了基础。在本文中，已探究了与常用于从卷心菜叶中分离的酶相对应的同工酶（PLD2）的三个序列特征在水解以及转磷脂酰化活性中的重要性：（i）两个HKD基序，（ii）C末端和（iii）八个半胱氨酸残基。所有这些区域或氨基酸在α型植物PLD中高度保守。基于多序列比对、二级结构预测和疏水性图谱比较，创建并测定了35种酶变体。所有测试位置在无甘油时对水解活性以及有甘油时对水解和转磷脂酰化活性的比例而言，对氨基酸交换都非常敏感。通过C310S和C625S替换可显著提高总活性和转磷脂酰化活性。
