Computational protocol: The Crystal Structure of D-Threonine Aldolase from Alcaligenes xylosoxidans Provides Insight into a Metal Ion Assisted PLP-Dependent Mechanism

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Protocol publication

[…] A complete diffraction dataset was collected from a single crystal at the Swiss Light Source (SLS) of the Paul Scherrer Institute in Villingen, Switzerland (beamline X06DA). A full dataset was collected from a monoclinic crystal (space group P21) up to 1.5 Å resolution and processed with the program AUTOMAR (marScale version 3.09–6, MarResearch). The calculated Matthews coefficient [,] indicated a 99% probability for the presence of two AxDTA molecules per asymmetric unit. The structure was determined by following the protocol of DiMaio et al. [] employing the programs PHASER [] and ROSETTA []. Suitable template structures were identified using the web server HHpred []. The highest-ranking models (PDB-entries 3GWQ and 3LLX) were used as initial molecular replacement templates for PHASER []. For the top five PHASER-solutions initial density maps were calculated and used as additional input for ROSETTA. Applying a 10% energy cutoff, ROSETTA built 2000 new models for each PHASER solution. To evaluate and score these models in terms of structural relevance, PHASER runs in MR_RNP mode (molecular replacement, refinement and phasing) were performed. The best scoring models (based on log-likelihood statistics) were further used as input for the automated chain-tracing/rebuilding programs AutoBuild [] and ArpWarp []. Both programs independently produced almost complete structures of both chains.Structure refinement and model rebuilding were carried out with the programs PHENIX [] and COOT [] by alternating real-space fitting against σA-weighted 2F O –F C and F O –F C electron density maps and least square optimizations. Rfree values were computed from 5% randomly chosen reflections, which were not used during refinement []. Clear residual electron density was assigned to the PLP-cofactor, one sodium ion and one manganese ion in each protomer. No electron density was observed for amino acids 1–6 in protomer A as well as for residues 1–7 in protomer B. Water molecules were placed into the difference electron density map and accepted or rejected according to geometry criteria as well as refined B-factors. In the later stages of the refinement, two TLS groups per protomer (including the respective PLP-cofactor) were defined based on an analysis using the TLSMD web server []. In addition, anisotropic atomic displacement parameters were refined for the four cations (2 Mn2+, 2 Na+). The final model was refined to R = 15% and Rfree = 18%. Validation of the structure was carried out with the program MOLPROBITY [] yielding a Ramachandran plot with 98.4% of the residues in favored, 1.5% in allowed and 0.1% in disallowed regions. Details pertaining to data statistics and structure refinement are listed in . [...] To model putative binding modes of different substrates in the active site of AxDTA the program AutoDock 4.0 [] was used as implemented in YASARA Structure []. Molecular models of the two diastereomers of D-phenylserine, (2R,3S)-phenylserine and (2R,3R)-phenylserine, covalently linked to the PLP cofactor (thereby representing the external aldimine) were generated and optimized in YASARA. The total net charge of each ligand was -2. The formal charge of the manganese ion was set to +2. Lys59 which binds PLP in the enzyme’s resting state was treated as uncharged. Position and orientation as well as torsion angles—except those involving atoms of the delocalized π-system of PLP—were allowed to vary. The docking was restricted to a 28x28x23 Å box around the center of the active site. Twenty independent simulation runs were performed on each ligand employing a genetic algorithm (population size 150, number of generations 22000). Structures with the lowest energy in each independent run were clustered with a root-mean-square-deviation tolerance of 1.5 Å and subsequently filtered based on the binding mode of the PLP-part of the ligand compared to the crystal structure. Molecular mechanics optimization was done within YASARA using the YASARA2 force field and employing the standard optimization protocol [–]. […]

Pipeline specifications

Software tools AutoDock, YASARA Structure
Application Protein interaction analysis
Organisms Achromobacter xylosoxidans
Chemicals Acetaldehyde, Alanine, Amino Acids, Glycine, Pyridoxal Phosphate