Insights into the structure of plant alpha-type phospholipase D.
Phospholipases D play an important role in the regulation of cellular processes in plants and mammals. Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs. Knowledge of phospholipase D structures, however, is widely restricted to sequence data. The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D. In this study, the isoenzyme form of phospholipase D from white cabbage (PLDalpha2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure. The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops. The far-UV CD-spectroscopic data indicate the presence of alpha-helical as well as beta-structural elements, with the latter being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10-20 mm can be confirmed. The stability of PLDalpha2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation.
Stumpe
Susanne
S
Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Halle, Saale, Germany.
König
Stephan
S
Ulbrich-Hofmann
Renate
R
D001937:Brassica / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D004795:Enzyme Stability; D008958:Models, Molecular; D009994:Osmolar Concentration; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D010739:Phospholipase D; D002118:Calcium