Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca 2+ )-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I 8 W 4 W 1 ). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca 2+ -saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca 2+ -CML19. Importantly, the peptide dramatically increases the affinity for Ca 2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca 2+ -saturated and bound to SAC3B even at resting Ca 2+ -levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca 2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.
Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5′-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro , suggesting a role in both arginine and GABA metabolism in vivo . The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention.
L -Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5´-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L -dopa and other L -aromatic amino acids. To advance structure–function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the latter protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the V max was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme–apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.