Proteolytic sensitivity of a recombinant phospholipase D from cabbage: identification of loop regions and conformational changes.
A recombinant phospholipase D from white cabbage (PLD2) composed of 812 amino acid residues was studied by site-directed mutagenesis and limited proteolysis to obtain first information on its tertiary structure. Limited proteolysis by thermolysin resulted in the formation of some large fragments of PLD2. From mass spectrometry and N-terminal sequencing of the peptides, the cleavage sites could be identified (1. Thr41-Ile42, 2. Asn323-Leu324 or Gly287-Leu288 and Ser319-Ile320 in case of the mutant L324S-PLD2). This suggested an exposed loop in the C2 domain of PLD2 and a large flexible region close to the N-terminal side of the first catalytic (HKD) motif. Calcium ions, the substrate 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the competitive inhibitor 1,3-dipalmitoylglycero-2-phosphocholine influenced the proteolytic cleavage. Calcium ions exerted a destabilizing effect on the conformation of PLD2.
Younus
H
H
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Schöps
R
R
Lerchner
A
A
Rücknagel
K P
KP
Schierhorn
A
A
Saleemuddin
M
M
Ulbrich-Hofmann
R
R
D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002918:Chymotrypsin / Q000378:metabolism; D006358:Hot Temperature; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012694:Serine / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D012694:Serine; D010739:Phospholipase D; D002918:Chymotrypsin; D014357:Trypsin; D013820:Thermolysin; D008239:Lysine; D002118:Calcium