Molecular rules for selectivity in lipase-catalysed acylation of lysine

International audienceThe selectivity of L-lysine acylation by lauric acid catalysed by Candida antarctica lipase B (CALB) was investigated combining experimental and theoretical methodologies. Experiments showed the near-exclusive acylation of lysine ε-amino group; only traces of product resulting from the acylation of lysine α-amino group were observed fleetingly. Molecular modelling simulations were performed aiming to understand the molecular rules for selectivity. Flexible docking simulations combined with structural investigations into lysine/CALB binding modes also suggested the preferential acylation of lysine ε-amino group without, however, excluding the acylation of the lysine α-amino group. Electrostatic interaction energy between lysine and the residues covering the catalytic cavity was calculated in order to understand the discrimination between the two lysine amino groups. The results suggests that the proximity of the carboxylate group hinders the binding of the substrate in configurations enabling the N-acylation. Key interactions with the polar region covering the catalytic triad were identified and a plausible explanation for selectivity was proposed

Pharmacological Modeling and Regulation of Excitatory Amino Acid Transporters (EAATs)

L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS that can mechanistically contribute to either neuronal signaling or neuronal pathology. Consequently, its concentration in the CNS must be carefully regulated, a critical need that is met by the excitatory amino acid transporters (EAATs). The presence of at least five isoforms of EAATs raises interesting questions as to potential structural and functional differences among the subtypes. We have investigated possible differences in the ligand binding domains of the EAATs through the development of computationally based pharmacophore models. An EAAT2-specific model was created with four potent and selective ligands that act as non-substrate inhibitors: cis-5-methyl-L-trans-2,3-pyrrolidine dicarboxylate, L-anti-endo-3,4-methano-pyrrolidine-3,4-dicarboxylate (L-anti-endo-3,4-MPDC), (2S,3R,4S)-2-(carboxy-cyclopropyl) glycine (L-CCG-IV) and L-B-threo-benzyloxy-aspartate (L-B-TBOA). This model predicts distinct regions that might influence the potency and selectivity of EAAT2 ligands, including: 1) a highly conserved positioning of the two carboxylate and the amino groups, 2) a nearby region that can accommodate selective modifications (e.g., cyclopropyl ring, CH3 groups, and O atoms), and 3) a region occupied by the benzyl ring of L-B-TBOA. This model was also used in conjunction with L-B-threo-benzyl aspartate (L-B-TBA), a recently characterized preferential inhibitor of EAAT3, to identify possible differences between EAAT2 and EAAT3. Functional studies on the EAATs also led to the identification of a putative modulatory mechanism that is specific for EAAT1. Thus, a series of sulfated neuroactive steroids, including pregnenolone sulfate (PREGS), were found to selectively increase the ability of EAAT1 to transport atypical substrates like D-aspartate and L-cysteine, but not L-glutamate. The effect was rapid, reversible, limited to a select group of sulfated steroids, and not observed with either EAAT2 or EAAT3. Interestingly, the action of PREGS could be blocked by the simultaneous addition of arachidonic acid, a previously recognized inhibitory modulator of EAAT1. The fact that this observed change in activity was produced by neurosteroids raises questions not only related to the regulatory mechanisms itself, but also to the possible role of neurosteroid in modulating glutamate transport