Molecular dynamics simulations and kinetic measurements provide insights into the structural requirements of substrate size-dependent specificity of oligogalacturonide oxidase 1 (OGOX1).

Oligogalacturonides (OGs) are pectin fragments released from the breakdown of the homogalacturonan during pathogenesis that act as Damage-Associated Molecular Patterns. OG-oxidase 1 (OGOX1) is an Arabidopsis berberine bridge enzyme-like (BBE-l) oligosaccharide oxidase that oxidizes OGs, impairing their elicitor activity and concomitantly releasing H2O2. The OG-oxidizing activity of OGOX1 is markedly pH-dependent, with optimum pH around 10, and is higher towards OGs with a degree of polymerization higher than two. Here, the molecular determinants of OGOX1 responsible for the binding of OGs with different lengths have been investigated through molecular dynamics simulations and enzyme kinetics studies. OGOX1 was simulated in complex with OGs with different degree of polymerization such as di-, tri-, tetra- and penta-galacturonide, in water solution at alkaline pH. Our simulations revealed that, among the four OGOX1/OG combinations, the penta-galacturonide (OG5) showed the best conformation in the active site to be efficiently oxidized by OGOX1. The optimal conformation can be stabilized by salt-bridges formed between the carboxyl groups of OG5 and five positively charged amino acids of OGOX1, highly conserved in all OGOX paralogs. Our results suggest that these interactions limit the mobility of OG5 as well as longer OGs, contributing to maintain the terminal monomer of OGs in the optimal orientation in order to be oxidized by the enzyme. In accordance with these results, the enzyme efficiency (Kcat/KM) of OGOX1 on OG5 (40.04) was found to be significantly higher than that on OG4 (13.05) and OG3 (0.6).

A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1.

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1.

Molecular dynamics of an asymmetric form of GabR, a bacterial transcriptional regulator.

GabR is a bacterial transcription regulator with a dimeric structure in which each subunit includes a wHTH (winged Helix-Turn-Helix) domain connected through a peptide linker to a large C-terminal domain folded as the enzyme aspartate aminotransferase (AAT). In Bacillus subtilis, GabR activates the genes involved in the metabolism of γ-amino butyric acid (GABA) upon formation of a PLP-GABA adduct. Recently, the crystallographic structure of an asymmetric form of GabR has been solved. This form (semi-holo) has one active site binding PLP as internal aldimine and the other the PLP-GABA complex. This work reports a molecular dynamics (MD) study aimed at understanding the characteristics of the asymmetric GabR form and compare them to the dynamics properties of previously studied symmetric holo (internal PLP aldimine at both active sites) and holo-GABA (containing PLP-GABA adducts) GabRs. Standard molecular dynamics and data analysis techniques have been used. The results indicate a remarkable asymmetry in the mobility and interactions of the different structural portions of the semi-holo GabR and of a few residues at the active site. The pattern is different from that observed in the other symmetrical GabR forms. The asymmetric perturbation of the active site residues may suggest the existence of a form of allosteric interference between the two active sites.

Conformational transitions induced by γ-amino butyrate binding in GabR, a bacterial transcriptional regulator.

GabR from Bacillus subtilis is a transcriptional regulator of the MocR subfamily of GntR regulators. The MocR architecture is characterized by the presence of an N-terminal winged-Helix-Turn-Helix domain and a C-terminal domain folded as the pyridoxal 5'-phosphate (PLP) dependent aspartate aminotransferase (AAT). The two domains are linked by a peptide bridge. GabR activates transcription of genes involved in γ-amino butyrate (GABA) degradation upon binding of PLP and GABA. This work is aimed at contributing to the understanding of the molecular mechanism underlying the GabR transcription activation upon GABA binding. To this purpose, the structure of the entire GabR dimer with GABA external aldimine (holo-GABA) has been reconstructed using available crystallographic data. The structure of the apo (without any ligand) and holo (with PLP) GabR forms have been derived from the holo-GABA. An extensive 1 µs comparative molecular dynamics (MD) has been applied to the three forms. Results showed that the presence of GABA external aldimine stiffens the GabR, stabilizes the AAT domain in the closed form and couples the AAT and HTH domains dynamics. Apo and holo GabR appear more flexible especially at the level of the HTH and linker portions and small AAT subdomain.