Mechanism of Action of Flavin-Dependent Halogenases

To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an −NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory “cluster” models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms

Preliminary assessment of the activity and stability of the phytase variants with engineered disulfide bridges.

<p>(A) Activity values for the wild-type and the variants studied at pH 4.5. (B) Profiles of activity vs. temperature at pH 4.5. The drop in activity at the higher temperatures indicates denaturation and provides a first estimate of the thermal stability. (C) Profiles of activity vs. pH for wild-type and the variants. In panels B and C, the maximum activity value of each profile is normalized to 100, while in panel A wild-type phytase is assigned a value of 100. Code color for the variants in panels B and C refers to the number of engineered bridges (black 0, blue 1, green 2, red 3) and is more clearly apparent in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone-0070013-g005" target="_blank">Figure 5A</a>.</p

Model used to derive equation 3 and describe the experimental thermal inactivation profiles for wild-type phytase and variants (Figures 7A and B).

<p>(A) An intermediate state (or ensemble) is assumed to be critical for the irreversible denaturation process. (B) and (C) At equilibrium, the population of I is always low, although it reaches a maximum roughly within the temperature range of the transition. When using a logarithmic scale (panel C) the shape of the population of I versus temperature profiles matches that of the ln(τ_1/2) versus temperature plots of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone-0070013-g007" target="_blank">Figure 7</a>, with the maximum of population of I corresponding to the minimum of τ_1/2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.e003" target="_blank"><b>equation 3</b></a>). The profiles in panels B and C have been calculated using equations provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.s005" target="_blank">Text S2</a>.</p

α‑Helix or β‑Turn? An Investigation into N‑Terminally Constrained Analogues of Glucagon-like Peptide 1 (GLP-1) and Exendin‑4

Peptide agonists acting on the glucagon-like peptide 1 receptor (GLP-1R) promote glucose-dependent insulin release and therefore represent important therapeutic agents for type 2 diabetes (T2D). Previous data indicated that an N-terminal type II β-turn motif might be an important feature for agonists acting on the GLP-1R. In contrast, recent publications reporting the structure of the full-length GLP-1R have shown the N-terminus of receptor-bound agonists in an α-helical conformation. To reconcile these conflicting results, we prepared N-terminally constrained analogues of glucagon-like peptide 1 (GLP-1) and exendin-4 and evaluated their receptor affinity and functionality <i>in vitro</i>; we then examined their crystal structures in complex with the extracellular domain of the GLP-1R and used molecular modeling and molecular dynamics simulations for further investigations. We report that the peptides’ N-termini in all determined crystal structures adopted a type II β-turn conformation, but <i>in vitro</i> potency varied several thousand-fold across the series. Potency correlated better with α-helicity in our computational model, although we have found that the energy barrier between the two mentioned conformations is low in our most potent analogues and the flexibility of the N-terminus is highlighted by the dynamics simulations

Structural consequences of an engineered disulfide crosslink.

<p>(A) The fold of Chain A of <i>C. braakii</i> phytase in ribbon format, with residues 6–18 in ice blue, 19–46 yellow, 47–135 blue, 136–259 red and 260–410 grey. The four conserved disulfide bridges are shown as cylinders, and the engineered ones as spheres. The phytate analogue myo-inositol hexakissulfate – shown as cylinders-has been modeled into the active site based on its position in its complex with <i>H. alvei</i> phytase (PDB 4aro). (B) The electron density in the final 2F_o–F_c synthesis contoured at the 1σ level around the engineered disulfide bridge between residues 141 and 199. (C) Superposition of the structures of the <i>E. coli</i> (green, PDB 1dkq) and <i>H. alvei</i> (coral, PDB 4ars) enzymes on <i>C. braakii</i> phytase (blue), using the SSM option <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013-Krissinel1" target="_blank">[44]</a> in CCP4mg. The structures are shown in worm and tube format. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone-0070013-g004" target="_blank">Figures 4</a> A–C were all made with CCP4mg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013-McNicholas1" target="_blank">[45]</a>.</p

Thermal inactivation kinetics data for wild-type phytase and variants.

<p>(A) and (B) Illustrative plots of activity versus time for experiments performed at several temperatures with (A) a variant with two engineered bridges and (B) wild-type. Total protein concentration is 0.5 mg/mL. The continuous lines represent the best fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.e001" target="_blank">equation 1</a> to the experimental data. The fastest kinetics are observed at an intermediate temperature (74°C in A and 69.5°C in (B). (C) Values of the reaction order derived from the fitting of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.e001" target="_blank">equation 1</a> to inactivation profiles for wild-type phytase and all the variants. (D) Agreement between the rate constants obtained with total protein concentration of 0.5 and 1 mg/mL. (E) Representative example of the protein concentration effect on the rate of irreversible denaturation (variant D1, 76°C).</p

Temperature dependence of the time scale (τ_1/2) for the irreversible denaturation of wild type phytase and variants.

<p>(A) and (B) Plots of ln(τ_1/2) versus temperature for the variants (for the sake of clarity, only wild type and the variant with three engineered bridges are included in panel A). The continuous lines represent the best fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.e003" target="_blank">equation 3</a> and the Inset in panel B is a plot of a measure of goodness of fit (χ²) versus the reaction order m in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.e003" target="_blank">equation 3</a> (see main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone.0070013.s005" target="_blank">Text S2</a> for details). Thicker lines indicate the temperature range of denaturation transition as seen by DSC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone-0070013-g005" target="_blank">Figure 5A</a>). The colors of the lines and data points refer to the variant, as specified in panel A of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070013#pone-0070013-g005" target="_blank">Figure 5</a>, with the number of engineered bridges indicated. (C) Enthalpies of the unfolded and intermediate states (relative to the native state) plotted versus the corresponding T₀ value. (D) Correlation between the effect of engineered disulfide bridges on the free energy of the unfolded and intermediate states (relative to the native state) at a temperature of 70°C.</p

Molecular Dynamics simulations performed on a homology model of the phytase from <i>C. braakii</i>.

<p>Simulations were performed at several temperatures (shown in Kelvin in the inset of the upper panel). Upper panel: isotropic root mean square deviations (iRMSF) of the Cα at the different temperatures. Middle panel: secondary structure assignment with red regions representing α-helices and yellow regions representing β-sheets. Bottom panel: mean distances between Cα of the starting structure and Cα of the structures at different temperatures. These profiles suggest that at high temperature the enzyme displays an unfolding behavior while at low temperatures the available thermal energy is only able to excite fluctuations of specific regions. Dashed grey lines mark the position of the residues mutated to engineer disulfide bridges.</p

Degradation of Phytate by the 6-Phytase from <i>Hafnia alvei</i>: A Combined Structural and Solution Study

<div><p>Phytases hydrolyse phytate (<i>myo</i>-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated <i>myo</i>-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, <i>Hafnia alvei</i> and <i>Yersinia kristensenii</i>, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the <i>H. alvei</i> enzyme in complex with the substrate analogue <i>myo</i>-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the <i>H. alvei</i> complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.</p></div