No abstract

Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate, whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity.

The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain-structural changes that ensure a hierarchical succession of events during protein targeting.

b-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoylCoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral speci®city. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid residues 618-736 of human MFE-2 (dÁhÁSCP-2L) was solved at 1.75 A Ê resolution in complex with Triton X-100, an analog of a lipid molecule. This is the ®rst reported structure of an MFE-2 domain. The dÁhÁSCP-2L has an a/b-fold consisting of ®ve b-strands and ®ve a-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of dÁhÁSCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solventexposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.