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

Abstract

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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.

Fig. 1.

Alignment of PLD sequences from Streptomyces. All of the PLDs except that from S. chromofuscus are Ca²⁺-independent. Residues in red are conserved in all PLDs; residues in blue are conserved among the Ca²⁺-independent PLDs.

Fig. 2.

Sequence of S. chromofuscus rPLD with differences from the previously published sequence for S. chromofuscus PLD indicated on the line below. N-PLD would have the first four residues as TTGT instead of ADQA.

Fig. 3.

SDS-PAGE showing overexpression and purification of rPLD from pET expression (A) and H226A from the IMPACT fusion protein expression system (B). (A): (lane 1) protein content of the crude cell extract, (lane 2) protein content after acetone precipitation, (lane 3) protein content after the palmitoyl-cellulose column, and (lane 4) protein content after QFF chromatography. Molecular masses for the standard proteins are 66, 45, 36, 29, 24, and 20.1 kD. (B): (lane 1) protein content of lysed cell extract, (lane 2) protein content of flow-through from the chitin column, (lane 3) protein content of wash from the column, and (lane 4) protein eluted after incubation of column with DTT.

Fig. 3.

SDS-PAGE showing overexpression and purification of rPLD from pET expression (A) and H226A from the IMPACT fusion protein expression system (B). (A): (lane 1) protein content of the crude cell extract, (lane 2) protein content after acetone precipitation, (lane 3) protein content after the palmitoyl-cellulose column, and (lane 4) protein content after QFF chromatography. Molecular masses for the standard proteins are 66, 45, 36, 29, 24, and 20.1 kD. (B): (lane 1) protein content of lysed cell extract, (lane 2) protein content of flow-through from the chitin column, (lane 3) protein content of wash from the column, and (lane 4) protein eluted after incubation of column with DTT.

Fig. 4.

Comparison of secondary structure for rPLD (solid bars), H187A (//// bars), H187N (shaded bars), H200A (\\\\ bars), and H200N (open bars) as estimated from the CD spectrum using ellipticities in the region 190–260 nm. The error bars on the elements for wild-type PLD represent standard deviations for multiple determinations of secondary structure using rPLD, M1-PLD, and N-PLD.

Fig. 5.

Intrinsic fluorescence intensity at 337 nm of PLD (24 μg/mL) as a function of added Ca²⁺: (open circles) rPLD; (filled circles) H226A; (filled triangles) H187N; and (filled squares) C123S.