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Cloning, overexpression, and characterization of a bacterial Ca²⁺-dependent phospholipase D

Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA

Reprint requests to: Mary F. Roberts, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA; e-mail: mary.roberts{at}bc.edu; fax: (617) 552-2705.

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca²⁺-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca²⁺-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4–5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca²⁺-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10^-5 and 10^-4. The Cys mutants still bound Ca²⁺, so that it is likely that this residue is part of the active site of the Ca²⁺-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.

Keywords: Phospholipase D; Streptomyces chromofuscus; HKD motif; Ca2+-dependent enzyme; mutagenesis

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