Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core.

Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. The 1.9 A resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with the N-termini of two alpha-helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl-CoA carboxylase, whose beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.

Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence.

The 12S subunit of transcarboxylase is a 338 000 Da hexamer that transfers carboxlylate from methylmalonyl-CoA (MM-CoA) to biotin; in turn, the biotin transfers the carboxylate to pyruvate on another subunit, the 5S. Here, Raman difference microscopy is used to study the binding of substrate and product, and their analogues, to single crystals of 12S. A single crystal is the medium of choice because it provides Raman data of unprecedented quality. Crystalline ligand-protein complexes were formed by cocrystallization or by the soaking in/soaking out method. Raman difference spectra were obtained by subtracting the spectrum of the apo crystal from that of a crystal with the substrate or product bound. Raman difference spectra from crystals with the substrate bound are dominated by bands from the protein's amide bonds and aromatic side chain residues. In contrast, Raman difference spectra involving the product, propionyl-CoA, are dominated by modes from the ligand. These results show that substrate binding triggers a conformational change in 12S, whereas product binding does not. The conformational change involves an increase in the amount of alpha-helix since markers for this secondary structure are prominent in the difference spectra of the substrate complex. The number of MM-CoA ligands bound per 12S hexamer can be gauged from the intensity of the MM-CoA Raman features and the fact that the protein concentration in the crystals is known from X-ray crystallographic data. Most crystal samples had six MM-CoAs per hexamer although a few, from different soaking experiments, contained only 1-2. However, both sets of crystals showed the same degree of protein conformational change, indicating that the change induced by the substrate is cooperative. This effect allowed us to record the Raman spectrum of bound MM-CoA without interference from protein modes; the Raman spectrum of a 12S crystal containing 2 MM-CoA ligands per hexamer was subtracted from the Raman spectrum of a 12S crystal containing six MM-CoA ligands per hexamer. The conformational change is reversible and can be controlled by soaking out or soaking in the ligand, using either concentrated ammonium sulfate solutions or the solution used in the crystallization trials. Malonyl-CoA also binds to 12S crystals and brings about conformational changes identical to those seen for MM-CoA; in addition, butyryl-CoA binds and behaves in a manner similar to propionyl-CoA. These data implicate the -COO- group on MM-CoA (that is transferred to biotin in the reaction on the intact enzyme) as the agent bringing about the cooperative conformational change in 12S.

Characterization of the carboxylate delivery module of transcarboxylase: following spontaneous decarboxylation of the 1.3S-CO2- subunit by NMR and FTIR spectroscopies.

Transcarboxylase (TC) is a multisubunit enzyme that catalyzes the transfer of a carboxylate group from methylmalonyl-CoA (MMCoA) to pyruvate. The CO2- group is shuttled between the MMCoA and pyruvate binding sites by a biotin cofactor, covalently linked to the 1.3S subunit. Fully carboxylated 1.3S can be prepared in vitro using 1.3S, MMCoA, and catalytic amounts of the TC's MMCoA-binding subunit. The 1.3S-CO2- intermediate decarboxylates spontaneously over a period of hours, and this process was characterized by 1D and 2D NMR and FTIR spectroscopies. The NMR data yielded a first-order kinetic constant of 1.4 x 10(-3) min(-1) for the spontaneous decarboxylation. This rate was calculated from the 1D NMR spectrum by measuring the reappearance of biotin's ureido NH protons and the disappearance of peaks at 6.99 and 7.67 ppm assigned to Asn-8 and/or Asn-24 from the 1.3S's N-terminus. The latter peaks are absent in the 1D spectrum of non-carboxylated 1.3S due to exchange between two or more conformations within the N-terminus causing line broadening. It is proposed that interactions between the biotin-CO2- and the N-terminal amino acids perturb this conformational equilibrium causing some N-terminal residues to appear in the 1D NMR spectrum of the carboxylated form. Further details are apparent from a comparison of the 2D spectra of the 1.3S-CO2- and 1.3S proteins, where carboxylation causes several peaks from the C-terminal half to shift as well as the appearance of resonances due to some residues located at the N-terminal half of the protein. FTIR difference spectra were used also to follow spontaneous decarboxylation of the 1.3S-CO2-. For the carboxylated 1.3S, the difference spectra provided the vibrational signature of the CO2- on the biotin ring. A doublet was identified at 1695 and 1699 cm(-1) that increased in intensity with increasing t. This is assigned to an antisymmetric stretching vibration of the CO2- group bound to biotin on the 1.3S protein. Its position and profile provide further evidence for interactions occurring between the biotin-CO2- group and the 1.3S protein. These studies demonstrate the highly mobile, "poised" nature of the 1.3S protein engineered for its role as a CO2- translocator.