Engineering the active site of ascorbate peroxidase.

E Lloyd Raven; A Celik; P M Cullis; R Sangar; M J Sutcliffe

doi:10.1042/0300-5127:0290105

Home/Archive/PubMed

PubMed · 11356136

Engineering the active site of ascorbate peroxidase.

E Lloyd Raven·A Celik·P M Cullis·R Sangar·M J Sutcliffe

View original source ↗Export metadata ↓

Abstract

Understanding the catalytic versatility of haem enzymes, and in particular the relationships that exist between different classes of haem-containing proteins and the mechanisms by which the apo-protein structure controls chemical reactivity, presents a major experimental and theoretical challenge. These issues are discussed in the general context of peroxidase and cytochrome P450 chemistry, and specific issues relating to the catalytic chemistry of ascorbate peroxidase are highlighted.

Explore related subjects

Ascorbate Peroxidases Binding Sites Cresols Cytochrome P-450 Enzyme System Heme Models, Molecular Oxidation-Reduction Peroxidases Protein Engineering Substrate Specificity Sulfides Journal Article Review

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

E Lloyd Raven, A Celik, P M Cullis, R Sangar, M J Sutcliffe. 2001. Engineering the active site of ascorbate peroxidase.. https://doi.org/10.1042/0300-5127%3A0290105

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death.

An increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H(2)O(2) during time, or directly with known amounts of H(2)O(2). Data presented here indicate that, in TBY-2 cells, a difference in H(2)O(2) level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H(2)O(2)-dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H(2)O(2) generation. Under a threshold value of H(2)O(2) overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD.

Ascorbate Peroxidases↗

PubMed2006

Biosynthesis of ascorbic acid in legume root nodules.

Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by L-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling.

Ascorbate Peroxidases↗

PubMed2006

Heme oxygenase up-regulation in ultraviolet-B irradiated soybean plants involves reactive oxygen species.

Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and leads to the generation of reactive oxygen species (ROS). Heme oxygenase (HO, EC 1.14.99.3) plays a protective role against oxidative stress in mammals, but little is known about this issue in plants. Here, we report for the first time the response of HO in leaves of soybean (Glycine max L.) plants subjected to UV-B radiation. Under 7.5 and 15 kJ m(-2 )UV-B doses, HO, catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were increased and the production of thiobarbituric acid reactive substances (TBARS) regain control values after 4 h of plant recuperation. Treatment with 30 kJ m(-2) UV-B provoked a decrease in these antioxidant enzyme activities. Immunoblot analysis showed a 4.3 and 3.7-fold increase in HO-1 protein expression after irradiation with 7.5 and 15 kJ m(-2), respectively. HO-1 transcript levels were enhanced (up to 77%) at these doses, as assessed by semi-quantitative RT-PCR. These data demonstrated that increased HO activity was associated with augmented protein expression and transcript levels. Plants pre-treated with the antioxidant ascorbic acid did not show the UV-B-induced up-regulation of HO-1 mRNA, but hydrogen peroxide treatment could mimic this reaction. Our results indicate that HO is up-regulated in a dose-depending manner as a mechanism of cell protection against oxidative damage and that such response occurred as a consequence of HO-1 mRNA enhancement involving ROS.

Ascorbate Peroxidases↗