Expression and characterization of a heterodimer of Streptomyces chromofuscus phospholipase D

Abstract

Streptomyces chromofuscus phospholipase D (PLD) is secreted by the bacterium and proteolytically cleaved to a more active form (PLD 37/18) where the two parts of the molecule are still tightly associated. Based on previous sequencing results of authentic PLD 37/18, we have constructed a vector consisting of separate ORFs for the N-terminal and C-terminal portions of S. chromofuscus PLD and overexpressed active heterodimeric PLD. Neither fragment cloned separately folded properly. The identity of each peptide was confirmed by peptide-mass fingerprinting with MALDI-TOF mass spectrometry. The recombinant complex had a specific activity about six times higher than that of the recombinant intact PLD enzyme and was no longer activated by phosphatidic acid (PA). Phosphotransferase activity, binding affinity to phospholipid vesicles, loss of product activation, pH profile and pH-related Ca 2+ activation and inhibition were comparable to authentic PLD 37/18 purified from S. chromofuscus growth medium. PLD 37 alone could also be isolated; the enzyme was active but not as stable as PLD 37/18. These experimental results strongly support the hypothesis that the C-terminal peptide is necessary for correct folding and insertion of catalytic metal ions. However, they suggest the ligands involved in Fe 3+ coordination must be altered upon cleavage of the protein. Asp389, in the C-terminal fragment, whose replacement impairs Fe 3+ binding to the protein, must be replaced by another ligand, since the N-terminal fragment, once folded, is active. In the process of cloning the two peptides, the complete signal sequence for this protein was also determined. The signal peptide of S. chromofuscus PLD enzyme contained a twin arginine motif suggesting that S. chromofuscus PLD, like Bacillus subtilis phoD, is most likely secreted by the TAT translocation pathway under the transcriptional control of the pho regulon.