Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae

The formation of hydrogen peroxide (H₂O₂) by the FAD-dependent α-glycerophosphate oxidase (GlpO), is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. (SspGlpO) is similar to the pneumococcal protein (SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO (MpGlpO), having <20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO we designate as Type II GlpOs. Here, the recombinant His-tagged MpGlpO structure is described at ~2.5 Å resolution, solved by molecular replacement using as a search model the Bordetella pertussis protein 3253 (Bp3253) a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of ~580 min⁻¹ while Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for α-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. This work provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.Keywords: drug design, flavoenzyme, protein evolution, GlpA, hydride transfe

Kinetic Mechanism of L-α-Glycerophosphate Oxidase from Mycoplasma pneumoniae

L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H₂O₂). The catalytic properties of the recombinant His₆-GlpO from Mycoplasma pneumoniae (His₆-MpGlpO) were investigated with transient and steady-state kinetics and ligand binding. The results indicate that the reaction mechanism of His₆-MpGlpO follows a ping-pong model. Double-mixing stopped-flow experiments show that after flavin-mediate substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values of the individual rate constants and k [subscript]cat (4.2 s⁻¹ at 4 °C) determined, in addition to the finding that H₂O₂ can bind to the oxidized enzyme suggest that H₂O₂ release is the rate-limiting step for the overall reaction. Results indicate that His₆-MpGlpO contains mixed populations of fast and slow reacting species. Only the fast reacting species predominantly participates in turnovers. Different from other GlpO enzymes previously reported, His₆-MpGlpO can catalyze the reverse reaction of reduced enzyme and DHAP. This result can be explained by the standard reduction potential value of His₆-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that DL-glyceraldehyde 3-phosphate (GAP) can be used as a substrate in the His₆-MpGlpO reaction, although it exhibited a ~100-fold lower k[subscript]cat value in comparison to the reaction of Glp. These results also imply the involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His₆-MpGlpO reported here should be useful for future studies of drug development against Mycoplasma pneumoniae infection

Creating Flavin Reductase Variants with Thermostable and Solvent‐Tolerant Properties by Rational‐Design Engineering

We have employed computational approaches—FireProt and FRESCO—to predict thermostable variants of the reductase component (C1) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6–5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH−) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300–500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications

Steady-state kinetics of PaDHPAO prepared in buffers in the absence or presence of ascorbic acid.

<p>The assay reactions containing 39 nM PaDHPAO and various concentrations of DHPA (0.5–2560 μM) were carried out in air-saturated (oxygen concentration ~260 μM) 50 mM sodium phosphate buffer at pH 7.5 and 25°C and the reactions were monitored by the absorption increase at 380 nm. Plots of the initial rates of PaDHPAO prepared in buffers in the presence (a solid line with filled diamonds) or absence (a dashed line with empty squares) of ascorbic acid <i>versus</i> concentration of DHPA are shown. All kinetic parameters of both conditions are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171135#pone.0171135.t006" target="_blank">Table 6</a>.</p

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from <i>Pseudomonas aeruginosa</i>: An Fe(II)-containing enzyme with fast turnover

<div><p>3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from <i>Pseudomonas aeruginosa</i> (PaDHPAO) was overexpressed in <i>Escherichia coli</i> and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM^-1cm^-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a <i>K</i>_m for DHPA of 58 ± 8 μM and a <i>k</i>_cat of 64 s^-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two p<i>K</i>_a values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(<i>k’</i>_cat) <i>versu</i>s the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (<i>E</i>_a) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.</p></div

¹H-NMR spectrum of the product from the PaDHAPO ring cleavage reaction.

<p>The product was isolated from the PaDHPAO reaction by ultrafiltration as described in the Methods section. The filtrate was lyophilized and resuspended in <i>d</i>_<i>6</i>- DMSO.</p

Inactivation of PaDHPAO by metal ions and redox active reagents.

<p>Inactivation of PaDHPAO by metal ions and redox active reagents.</p

Sequence similarity networks (SSN) for PaDHPAO constructed by EFI alignment score of 60 (~34% identity) illustrating 12 iso-function clusters of enzymes.

<p>Enzymes that were experimentally investigated are highlighted in color. PaDHPAO (this study) is yellow, <i>E</i>. <i>coli</i> C (Uniprot:Q05353) is orange, <i>K</i>. <i>pneumoniae</i> (Uniprot:Q9RE15) is red, <i>Pseudomonas</i> sp. (Uniprot:O33477) is green, and <i>C</i>. <i>testosteroni</i> (Uniprot:Q6J1Z6) is pink.</p

Preservation of the activated PaDHPAO activity.

<p>(A) Percentage of relative activity of the activated PaDHPAO decreased after the solution was kept on ice for 7–8 hours in 50 mM potassium phosphate buffer, pH 7.0. (B) Percentage of relative activity of the activated PaDHPAO in 50 mM potassium phosphate buffer, pH 7.0 containing different agents: (1) without any agents <i>(black)</i>, (2) 0.5 mM Fe(NH₄)₂(SO₄)₂ <i>(purple)</i>, (3) 1 mM DTT <i>(dark blue)</i>, (4) 0.5 mM ascorbic acid <i>(light blue)</i>, (5) 0.5 mM ascorbic acid and 1 mM DTT <i>(green)</i>, (6) 0.5 mM ascorbic acid, 1 mM DTT and 0.5 mM Fe(NH₄)₂(SO₄)₂ <i>(yellow)</i>, (7) 1 mM DTT and 0.5 mM Fe(NH₄)₂(SO₄)₂ <i>(orange)</i>, and (8) 0.5 mM ascorbic acid and 0.5 mM Fe(NH₄)₂(SO₄)₂ <i>(red)</i>. (C) Percentage of relative activity of the activated PaDHPAO in 50 mM potassium phosphate buffer, pH 7.0 containing different agents: (1) without any agents <i>(black)</i>, (2) 0.5 mM ascorbic acid <i>(cyan)</i>, (3) 1%(w/v) catalase <i>(pink)</i>, (4) 50 U/mL superoxide dismutase <i>(turquoise)</i> and (5) 1%(w/v) catalase and 50 U/mL superoxide dismutase <i>(brown)</i>. The results indicate that the highest level of % relative activity (around 90% up to ~ 6 hours) of the activated PaDHPAO can be achieved in the presence of 0.5 mM ascorbic acid <i>(cyan)</i>,catalase <i>(pink)</i> or superoxide dismutase <i>(turquoise)</i>).</p

Summary of biochemical and kinetic properties of PaDHPAO.

<p>Summary of biochemical and kinetic properties of PaDHPAO.</p