skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Activity of Cytosolic Ascorbate Peroxidase (APX) from Panicum virgatum against Ascorbate and Phenylpropanoids
APX is a key antioxidant enzyme in higher plants, scavenging H2O2 with ascorbate in several cellular compartments. Here, we report the crystal structures of cytosolic ascorbate peroxidase from switchgrass (Panicum virgatum L., Pvi), a strategic feedstock plant with several end uses. The overall structure of PviAPX was similar to the structures of other APX family members, with a bound ascorbate molecule at the ɣ-heme edge pocket as in other APXs. Our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Significantly, our study suggested that PviAPX can oxidize a broad range of phenylpropanoids with δ-meso site in a rather similar efficiency, which reflects its role in the fortification of cell walls in response to insect feeding. Based on detailed structural and kinetic analyses and molecular docking, as well as that of closely related APX enzymes, the critical residues in each substrate-binding site of PviAPX are proposed. Taken together, these observations shed new light on the function and catalysis of PviAPX, and potentially benefit efforts improve plant health and biomass quality in bioenergy and forage crops.  more » « less
Award ID(s):
2043248
PAR ID:
10475460
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
Int. J. Mol. Sci.
Date Published:
Journal Name:
International Journal of Molecular Sciences
Volume:
24
Issue:
2
ISSN:
1422-0067
Page Range / eLocation ID:
1778
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; (, Plant Physiology)
    Abstract In planta, H2O2 is produced as a by-product of enzymatic reactions and during defense responses. Ascorbate peroxidase (APX) is a key enzyme involved in scavenging cytotoxic H2O2. Here, we report the crystal structure of cytosolic APX from sorghum (Sorghum bicolor) (Sobic.001G410200). While the overall structure of SbAPX was similar to that of other APXs, SbAPX uniquely displayed four bound ascorbates rather than one. In addition to the ɣ-heme pocket identified in other APXs, ascorbates were bound at the δ-meso and two solvent-exposed pockets. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the ɣ-heme edge and δ-meso sites. Based on crystal structures, steady-state kinetics, and site-directed mutagenesis results, both ascorbate molecules at the ɣ-heme edge and the one at the surface are expected to participate in the oxidation reaction. We provide evidence that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid at the ɣ-heme edge, indicating two successive electron transfers from a single-bound ascorbate. In addition, the δ-meso site was shared with several organic compounds, including p-coumaric acid and other phenylpropanoids, for the potential radicalization reaction. Site-directed mutagenesis of the critical residue at the ɣ-heme edge (R172A) only partially reduced polymerization activity. Thus, APX removes stress-generated H2O2 with ascorbates, and also uses this same H2O2 to potentially fortify cell walls via oxidative polymerization of phenylpropanoids in response to stress. 
    more » « less
  2. ; ; ; ; ; ; ; ; (, Plant Physiology)
    null (Ed.)
    Abstract Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs. 
    more » « less
  3. ; ; ; ; ; ; ; ; (, Biomaterials Science)
    null (Ed.)
    Lack of long-term patency has hindered the clinical use of small-diameter prosthetic vascular grafts with the majority of these failures due to the development of neointimal hyperplasia. Previous studies by our laboratory revealed that small-diameter expanded polytetrafluoroethylene (ePTFE) grafts coated with antioxidant elastomers are a promising localized therapy to inhibit neointimal hyperplasia. This work is focused on the development of poly(diol- co -citrate- co -ascorbate) (POCA) elastomers with tunable properties for coating ePTFE vascular grafts. A bioactive POCA elastomer (@20 : 20 : 8, [citrate] : [diol] : [ascorbate]) coating was applied on a 1.5 mm diameter ePTFE vascular graft as the most promising therapeutic candidate for reducing neointimal hyperplasia. Surface ascorbate density on the POCA elastomer was increased to 67.5 ± 7.3 ng mg −1 cm −2 . The mechanical, antioxidant, biodegradable, and biocompatible properties of POCA demonstrated desirable performance for in vivo use, inhibiting human aortic smooth muscle cell proliferation, while supporting human aortic endothelial cells. POCA elastomer coating number was adjusted by a modified spin-coating method to prepare small-diameter ePTFE vascular grafts similar to natural vessels. A significant reduction in neointimal hyperplasia was observed after implanting POCA-coated ePTFE vascular grafts in a guinea pig aortic interposition bypass graft model. POCA elastomer thus offers a new avenue that shows promise for use in vascular engineering to improve long-term patency rates by coating small-diameter ePTFE vascular grafts. 
    more » « less
  4. ; (, Plasma)
    A cold atmospheric-pressure He-plasma jet (CAPPJ) interacts with air and water, producing reactive oxygen and nitrogen species (RONS), including biologically active ions, radicals, and molecules such as NOx, H2O2, HNO3, HNO2, and O3. These compounds can activate interfacial redox processes in biological tissues. The CAPPJ can oxidize N2 to HNO3 and water to H2O2 at the interface between plasma and water. It can also induce the oxidation of water-soluble redox compounds in various organisms and in vitro. This includes salicylic acid, hydroquinone, and mixtures of antioxidants such as L (+)-ascorbic acid sodium salt with NADPH. It can react with redox indicators, such as ferroin, in a three-phase system consisting of air, CAPPJ, and water. Without reducing agents in the water, the CAPPJ will oxidize the water and decrease the pH of the solution. When antioxidants such as ascorbate, 1,4-hydroquinone, or NADPH are present in the aqueous phase, the CAPPJ oxidizes these substances first and then oxidizes water to H2O2. The multielectron mechanisms of the redox reactions in the plasma-air/water interfacial area are discussed and analyzed. 
    more » « less
  5. ; ; (, TheoretiCS)
    In the Continuous Steiner Tree problem (CST), we are given as input a set of points (called terminals) in a metric space and ask for the minimum-cost tree connecting them. Additional points (called Steiner points) from the metric space can be introduced as nodes in the solution. In the Discrete Steiner Tree problem (DST), we are given in addition to the terminals, a set of facilities, and any solution tree connecting the terminals can only contain the Steiner points from this set of facilities. Trevisan [SICOMP'00] showed that CST and DST are APX-hard when the input lies in the $$\ell_1$$-metric (and Hamming metric). Chleb\'ik and Chleb\'ikov\'a [TCS'08] showed that DST is NP-hard to approximate to factor of $$96/95\approx 1.01$$ in the graph metric (and consequently $$\ell_\infty$$-metric). Prior to this work, it was unclear if CST and DST are APX-hard in essentially every other popular metric. In this work, we prove that DST is APX-hard in every $$\ell_p$$-metric. We also prove that CST is APX-hard in the $$\ell_{\infty}$$-metric. Finally, we relate CST and DST, showing a general reduction from CST to DST in $$\ell_p$$-metrics. Comment: Abstract shortened. Journal version for TheoretiCS 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email