(R)-3-hydroxybutyrate dehydrogenase: Selective phosphatidylcholine bindingby the C-terminal domain

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondria l enzyme that has a specific requirement of phosphatidylcholine (PC) for fu nction. 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 pr otein, 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 G ST) form well-defined protein-lipid complexes with either PC or phosphatidy lethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with diga lactosyl diglyceride vesicles) as demonstrated by flotation in sucrose grad ients. 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 s alt treatment. Thrombin cleavage of GST-CTBDH, either before or after recon stitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrome try) which retains lipid binding without attached GST, The BDH activator, 1 -palmitoyl-2-(1-pyreyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phosphol ipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectiv ely) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH wit h effective Stern-Volmer quenching constants, (K-Q)(eff), of 11 and 9.3 (%) (-1), respectively (half-maximal quenching at similar to 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyr enyl-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 bu t with similar to 15-fold lower (K-Q)(eff) (half-maximal quenching at simil ar to 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE w ith HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a P C-selective lipid binding domain of the PC-requiring BDH.