Streptococcus pneumoniae is not only a commensal of the nasopharyngeal epithelium, but may also cause life-threatening diseases. Immune-electron microscopy studies revealed that the bacterial glycolytic enzyme, phosphoglycerate kinase (PGK), is localised on the pneumococcal surface of both capsulated and non-capsulated strains and colocalises with plasminogen. Since pneumococci may concentrate host plasminogen (PLG) together with its activators on the bacterial cell surface to facilitate the formation of plasmin, the involvement of PGK in this process was studied. Specific binding of human or murine PLG to strain-independent PGK was documented, and surface plasmon resonance analyses indicated a high affinity interaction with the kringle domains 1–4 of PLG. Crystal structure determination of pneumococcal PGK together with peptide array analysis revealed localisation of PLG-binding site in the N-terminal region and provided structural motifs for the interaction with PLG. Based on structural analysis data, a potential interaction of PGK with tissue plasminogen activator (tPA) was proposed and experimentally confirmed by binding studies, plasmin activity assays and thrombus degradation analyses.

An efficient chemoselective method for the creation of semisynthetic lipases by site-specific incorporation of tailor-made peptides on the lipase-lid site was developed. These new enzymes showed excellent improved specificity and regio- or enantioselectivity in different biotransformations.

Ten novel taxanes bearing modifications at the C2 and C13 positions of the baccatin core have been synthesized and their binding affinities for mammalian tubulin have been experimentally measured. The design strategy was guided by (i) calculation of interaction energy maps with carbon, nitrogen and oxygen probes within the taxane-binding site of β-tubulin, and (ii) the prospective use of a structure-based QSAR (COMBINE) model derived from an earlier series comprising 47 congeneric taxanes. The tubulin-binding affinity displayed by one of the new compounds (CTX63) proved to be higher than that of docetaxel, and an updated COMBINE model provided a good correlation between the experimental binding free energies and a set of weighted residue-based ligand–receptor interaction energies for 54 out of the 57 compounds studied. The remaining three outliers from the original training series have in common a large unfavourable entropic contribution to the binding free energy that we attribute to taxane preorganization in aqueous solution in a conformation different from that compatible with tubulin binding. Support for this proposal was obtained from solution NMR experiments and molecular dynamics simulations in explicit water. Our results shed additional light on the determinants of tubulin-binding affinity for this important class of antitumour agents and pave the way for further rational structural modifications.

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg⁻¹) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg⁻¹), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.