Insight into the transition between the open and closed conformations of Thermus thermophilus carboxypeptidase.

Carboxypeptidase cleaves the C-terminal amino acid residue from proteins and peptides. Here, we report the functional and structural characterizations of carboxypeptidase belonging to the M32 family from the thermophilic bacterium Thermus thermophilus HB8 (TthCP). TthCP exhibits a relatively broad specificity for both hydrophilic (neutral and basic) and hydrophobic (aliphatic and aromatic) residues at the C-terminus and shows optimal activity in the temperature range of 75-80 °C and in the pH range of 6.8-7.2. Enzyme activity was significantly enhanced by cobalt or cadmium and was moderately inhibited by Tris at 25 °C. We also determined the crystal structure of TthCP at 2.6 Å resolution. Two dimer types of TthCP are present in the crystal. One type consists of two subunits in different states, open and closed, with a Cα RMSD value of 2.2 Å; the other type consists of two subunits in the same open state. This structure enables us to compare the open and closed states of an M32 carboxypeptidase. The TthCP subunit can be divided into two domains, L and S, which are separated by a substrate-binding groove. The L and S domains in the open state are almost identical to those in the closed state, with Cα RMSD values of 0.84 and 0.53 Å, respectively, suggesting that the transition between the open and closed states proceeds with a large hinge-bending motion. The superimposition between the closed states of TthCP and BsuCP, another M32 family member, revealed that most putative substrate-binding residues in the grooves are oriented in the same direction.

Crystallization and preliminary X-ray analysis of carboxypeptidase 1 from Thermus thermophilus.

Carboxypeptidase 1 from the thermophilic eubacterium Thermus thermophilus (TthCP1, 58 kDa), a member of the M32 family of metallocarboxypeptidases, was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals diffracted X-rays to beyond 2.6 A resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 171.0, b = 231.6, c = 124.9 A. The crystal contains three molecules in an asymmetric unit (VM = 2.11 A3 Da(-1)) and has a solvent content of 61.5%.