Control of C4a-Hydroperoxyflavin Protonation in the Oxygenase Component of <i>p</i>‑Hydroxyphenylacetate-3-hydroxylase

The protonation status of the peroxide moiety in C4a-(hydro)­peroxyflavin of <i>p</i>-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The p<i>K</i>_a for the wild-type (WT) enzyme is 9.8 ± 0.2, while the values for the H396N, H396V, and H396A variants are 9.3 ± 0.1, 7.3 ± 0.2, and 7.1 ± 0.2, respectively. The hydroxylation efficiency of these mutants is lower than that of the WT enzyme. Solvent kinetic isotope effect studies indicate that proton transfer is not the rate-limiting step in the formation of C4a-OOH. All data suggest that His396 may act as an instantaneous proton provider for the proton-coupled electron transfer that occurs before the transition state of C4a-OOH formation

Summary of biochemical and kinetic properties of PaDHPAO.

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

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

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

Molecular mass of purified PaDHPAO.

<p>(A) SDS-PAGE (12%(w/v)) of the purified PaDHPAO. Lane 1 is a protein molecular weight standard marker (kDa) (Enzmart Biotech, Thailand) and lane 2 is an enzyme solution after purification by Phenyl-Sepharose chromatography. (B) A plot of relative volumes (V_e/V_o) <i>versus</i> the logarithms of the known molecular masses of protein standards (□): (1) ferritin (440 kDa), (2) aldolase (158 kDa), (3) BSA (65.4 kDa), (4) ovalbumin (48.9 kDa), (5) chymotrypsinogen (22.8 kDa), (6) ribonuclease (15.8 kDa), and PaDHPAO (●).</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

An extradiol cleavage of DHPA by DHPAO.

<p>An extradiol cleavage of DHPA by DHPAO.</p

Summary of biochemical and catalytic properties of DHPAO from various organisms.

<p>Summary of biochemical and catalytic properties of DHPAO from various organisms.</p