This paper reports the structure of the double mutant Asp251Gly/Gln307His (here called A2), generated by random mutagenesis, is able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. Here, we report the crystal structure of the heme domain of the mutant in the substrate-free form and in complex with the substrate N-palmitoylglycine, together with its impact on thermal stability, reduction potential and electron transfer.The substrate-free structure adopts a conformation more similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop, part of the substrate access channel. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on Ihelix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules.Differential scanning calorimetry shows a population destabilized by 2.2°C compared to the wild type protein, probably reflecting the increased flexibility of part of the protein compared to WT. Moreover, a shift of the heme reduction potential by 50 mV toward positive values, a 2-folds higher first electron transfer rate in the absence of oxygen and a 4-folds higher NADPH consumption rate in the presence of oxygen as the only electron acceptors, when compared to wild type protein.The data demonstrate that a single mutation far away from the active site is able to trigger an increase in protein flexibility in a key region of the substrate access channel, shifting the conformational equilibrium toward the closed form of the protein that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.

Aromatase catalyzes the biosynthesis of estrogens from androgens. Owing to the physiological importance of this conversion of lipophilic substrates, the interaction with the lipid bilayer for this cytochrome P450 is crucial for its dynamics that must allow an easy access to substrates and inhibitors. Here, the aromatase-anastrozole interaction is studied by combining computational methods to identify possible access/egress routes with the protein inserted in the membrane and experimental tools aimed at the investigation of the effect of the inhibitor on the protein conformation. By means of molecular dynamics simulations of the protein inserted in the membrane, two channels, not detected in the starting crystal structure, are found after a 20-nSec simulation. Trypsin digestion on the recombinant protein shows that the enzyme is strongly protected by the presence of the substrate and even more by the inhibitor. DSC experiments show an increase in the melting temperature of the protein in complex with the substrate (49.3 °C) and the inhibitor (58.7 °C) compared to the ligand-free enzyme (45.9 °C), consistent with a decrease of flexibility of the protein. The inhibitor anastrozole enters the active site of the protein through a channel different from that used from the substrate and promotes a conformational change that stiffens the protein conformation and decreases the protein-protein interaction between different aromatase molecules.

The Saccaromices cerevisiae D-serine dehydratase is a pyridoxal 5'-phosphate dependent enzyme that requires zinc for its function. It catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. In this work, a new methodology for monitoring D-serine is presented. Our results show that this enzyme could be successfully used as a biological probe for detection of D-serine via fluorescence spectroscopy.

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