(R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain.
(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST. The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH. The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH.
Loeb-Hennard
C
C
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA.
McIntyre
J O
JO
D005456:Fluorescent Dyes / Q000737:chemistry; D005982:Glutathione Transferase / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000737:chemistry; D014364:Tryptophan
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D006639:Histidine; C021248:1-palmitoyl-2-pyrenedecanoylphosphatidylcholine; D014364:Tryptophan; D006884:Hydroxybutyrate Dehydrogenase; D005982:Glutathione Transferase; D013917:Thrombin