http://www.cnr.it/ontology/cnr/individuo/prodotto/ID17432
On the effects of site-specific mutations on activity and expression of the Streptomyces PMF phospholipase D (Articolo in rivista)
- Type
- Label
- On the effects of site-specific mutations on activity and expression of the Streptomyces PMF phospholipase D (Articolo in rivista) (literal)
- Anno
- 2006-01-01T00:00:00+01:00 (literal)
- Alternative label
Fedeli C., Carrea G., Monti D. (2006)
On the effects of site-specific mutations on activity and expression of the Streptomyces PMF phospholipase D
in Journal of molecular catalysis. B, Enzymatic (Print)
(literal)
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- Fedeli C., Carrea G., Monti D. (literal)
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- ISI Web of Science (WOS) (literal)
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- Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milan, Italy (literal)
- Titolo
- On the effects of site-specific mutations on activity and expression of the Streptomyces PMF phospholipase D (literal)
- Abstract
- 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. (literal)
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