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Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
Expression and characterization of a heterodimer of Streptomyces chromofuscus phospholipase D
Hongying Yang1, Mary F Roberts
1Merkert Chemistry Center, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02167, USA.
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.
Insights
Researchers engineered a more active Streptomyces chromofuscus phospholipase D (PLD) by separating its N-terminal and C-terminal portions. This active heterodimeric PLD shows enhanced specific activity and stability, revealing insights into enzyme folding and metal ion coordination.
Area of Science:
- Enzymology
- Protein Biochemistry
- Microbial Physiology
Background:
- Streptomyces chromofuscus secretes phospholipase D (PLD), which is proteolytically cleaved into a more active heterodimeric form (PLD37/18).
- Understanding the structural and functional roles of these cleaved domains is crucial for enzyme engineering and biotechnological applications.
Purpose of the Study:
- To construct and overexpress an active heterodimeric S. chromofuscus PLD from separate N-terminal and C-terminal open reading frames (ORFs).
- To investigate the contribution of each peptide fragment to enzyme activity, stability, and folding.
- To elucidate the mechanism of PLD secretion and metal ion coordination.
Main Methods:
- Construction of a vector with separate ORFs for S. chromofuscus PLD N-terminal and C-terminal portions.
- Overexpression and purification of the recombinant heterodimeric PLD complex.
- Peptide-mass fingerprinting using MALDI-TOF mass spectrometry to confirm peptide identity.
- Biochemical characterization including specific activity assays, pH profiles, and metal ion binding studies.
Main Results:
- Neither N-terminal nor C-terminal fragment, when cloned separately, folded correctly, indicating a requirement for heterodimerization.
- The recombinant heterodimeric PLD exhibited approximately six times higher specific activity than the intact recombinant PLD and was not activated by phosphatidic acid (PA).
- The C-terminal peptide is essential for proper folding and catalytic metal ion insertion, with Asp389 playing a key role in Fe(3+) coordination.
Conclusions:
- The C-terminal peptide of S. chromofuscus PLD is indispensable for correct protein folding and the insertion of catalytic metal ions.
- Cleavage of the protein alters the ligands involved in Fe(3+) coordination, suggesting a dynamic structural change.
- The signal peptide contains a twin arginine motif, indicating secretion via the TAT translocation pathway under pho regulon control.
