On the effects of site-specific mutations on activity and expression of the Streptomyces PMF phospholipase D

The effects of specific mutations on the hydrolytic activity and heterologous expression levels of Streptomyces PMF phospholipase D (PLD-PMF), an enzyme industrially employed for glycerophospholipid (GPL) modification, have been studied by using natural GPLs and synthetic chromogenic derivatives (p-nitrophenyl derivatives) as substrates. Specifically, the essential role of the histidines (H167 and H440) and lysines (K169 and K442) of the two highly conserved HXKX 4 DX 6 G(G/S)X(D/N) (HKD) domains for catalysis was shown by isolation and characteriza- tion of the corresponding enzyme variants, whose activity was completely lost without any significant structural modification. The abolishment of the phospholipase D (PLD) activity strongly influenced the expression levels in Escherichia coli, with recovery yields 5-10 times higher than with the wild-type enzyme, thus indicating that citotoxicity for this host is indeed strictly correlated to enzyme activity. On the contrary, enzyme variants where the conserved aspartic acids (D174 and D447) of the two HKD domains were replaced with asparagine residues showed a decreased but still detectable PLD activity. The hypothesis that these conserved residues might play a structural role is supported by evidence of net superficial charge modifications observed during enzyme variants purification.