Essential Role of Tyrosine 229 of the Oxaloacetate Decarboxylase β-Subunit in the Energy Coupling Mechanism of the Na⁺ Pump^†

The membrane-bound β-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na+ translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na+ binding, because in the Y229F orY229A mutants, the β-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a βC291E mutation was unstable in the absence of Na+ and dissociated into an α−γ subcomplex and the β-subunit. The enzyme could only be isolated in the presence of 0.5 M NaCl. These results are consistent with the notion that the β-subunit changes its conformation upon Na+ binding

NMR Structure of the Sterol Carrier Protein-2Implications for the Biological Role

The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone 15 N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for Investigations of the mode of action of SCP2. The SCP2 fold is formed by a five-stranded β-sheet and four α-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the β-sheet, which are all located in the polypeptide segment 8-102, was identified with the use of the spinlabeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding. (C) 2000 Academic Press

Crystallization and Initial X-Ray Analysis of Rabbit Mature Sterol Carrier Protein 2

Sterol carrier protein 2 (SCP2) is a basic intracellular protein which facilitates the in vitro intermembrane transfer of cholesterol, phospholipids and glycolipids. SCP2 was expressed in Escherichia coli, purified to apparent electrophoretic homogeneity and crystallized. Single crystals were obtained by hanging-drop vapour diffusion using ammonium sulfate as precipitant. These crystals belong to space group P41212 or its enantiomorph, with unit-cell parameters a = b = 57.5, c = 86.5 Å, and have one molecule in the crystallographic asymmetric unit. Intensity data to 1.8 Å resolution were collected from native SCP2 crystals using synchrotron radiation, were processed and scaled with an R(linear) = 4.9%