Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa

Bernardes, Amanda; Textor, Larissa C.; Santos, Jademilson Celestino dos; Cuadrado, Nazaret Hidalgo; Kostetsky, E. Ya.; Roig, Manuel G.; Bavro, Vassiliy N.; Muniz, J.R.C.; Shnyrov, Valery L.; Polikarpov, Igor

doi:10.1016/j.biochi.2015.01.014

Cited by 22 publications

(18 citation statements)

References 62 publications

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“…For example, a plant peroxidase isolated from Chamaerops excelsa palm tree (CEP) with very strong pH and thermal stability also possesses significant substrate inhibition kinetic behavior, which possibly can be attributed to the existence of two substrate binding sites. 47,48 Current work extends structural studies that suggest that the substrates 2,4,6-TCP and 2,4,6-TBP inhibit DHP's peroxidase function by substrate inhibition at high substrate concentration, which prevents the collapse of the protein stability during the catalytic reaction at high substrate concentration. In conclusion, substrate inhibition of the 2,4,6-TCP and 2,4,6-TBP distinguish the peroxidase function of DHP from that of HRP.…”

Section: The Journal Of Physical Chemistry Bmentioning

confidence: 82%

Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase

Zhao

Franzen

2015

J. Phys. Chem. B

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The time-resolved kinetics of substrate oxidation and cosubstrate H2O2 reduction by dehaloperoxidase-hemoglobin (DHP) on a seconds-to-minutes time scale was analyzed for peroxidase substrates 2,4,6-tribromophenol (2,4,6-TBP), 2,4,6-trichlorophenol (2,4,6-TCP), and ABTS. Substrates 2,4,6-TBP and 2,4,6-TCP show substrate inhibition at high concentration due to the internal binding at the distal pocket of DHP, whereas ABTS does not show substrate inhibition at any concentration. The data are consistent with an external binding site for the substrates with an internal substrate inhibitor binding site for 2,4,6-TBP and 2,4,6-TCP. We have also compared the kinetic behavior of horseradish peroxidase (HRP) in terms of kcat, Km(AH2) and Km(H2O2) using the same kinetic scheme. Unlike DHP, HRP does not exhibit any measurable substrate inhibition, consistent with substrate binding at the edge of heme near the protein surface at all substrate concentrations. The binding of substrates and their interactions with the heme iron were further compared between DHP and HRP using a competitive fluoride binding experiment, which provides a method for quantitative measurement of internal association constants associated with substrate inhibition. These experiments show the regulatory role of an internal substrate binding site in DHP from both a kinetic and competitive ligand binding perspective. The interaction of DHP with substrates as a result of internal binding actually stabilizes that protein and permits DHP to function under conditions that denature HRP. As a consequence, DHP is a tortoise, a slow but steady enzyme that wins the evolutionary race against the HRP-type of peroxidase, which is a hare, initially rapid, but flawed for this application because of the protein denaturation under the conditions of the experiment.

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Section: The Journal Of Physical Chemistry Bmentioning

confidence: 82%

Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase

Zhao

Franzen

2015

J. Phys. Chem. B

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“…Similar studies addressing kinetic parameters and microscopic rate constants carried out with African [26], Royal [19] and CEP [20,23] palm tree peroxidases have revealed that these enzymes exhibit greater reactivity towards ferulic acid and ABTS, followed by the aromatic amines o-dianisidine, o-phenylendiamine and, finally, by phenolic substrates with one or two hydroxyl groups in their chemical structures. In contrast, both soybean and peanut peroxidases are more reactive towards guaiacol than towards amines [26][27][28].…”

Section: Ping-pong Bi-bi Microscopic Rate Constants For Cmpmentioning

confidence: 79%

“…Peroxidase activity [18][19][20][21][22][23] towards guaiacol was measured spectrophotometrically at 25 • C. An aliquot of enzyme solution was added to a 1-cm optical path-length spectral cuvette containing 18 mM guaiacol and 4.9 mM H 2 O 2 in 20 mM sodium phosphate buffer, pH 6.0, in a final volume of 2 mL. The rate of change in absorbance due to substrate oxidation was monitored at 470 nm.…”

Section: Enzyme Activitymentioning

confidence: 99%

Kinetics of Spanish broom peroxidase obeys a Ping-Pong Bi–Bi mechanism with competitive inhibition by substrates

Galende

Cuadrado

Kostetsky

et al. 2015

International Journal of Biological Macromolecules

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“…Even in 6.8 M GdnHCl at 37 °C, the half-life for protein denaturation was 5 h. The only other non-hyperthermophilic heme proteins of which we are aware that are approximately as stable as HtaA-CR2 are the Class III plant peroxidases [73, 74]. For example, soybean peroxidase has a half-life for denaturation of 3.3 h at 25 °C in 6.8 M GdnHCl [73].…”

Section: Discussionmentioning

confidence: 99%

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae

Uluisik

Akbas

Lukat-Rodgers

et al. 2017

Journal of Inorganic Biochemistry

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HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV-visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤ 5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8 M GdnHCl at 37 °C, the half-life was 5 h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.

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Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa

Cited by 22 publications

References 62 publications

Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase

Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase

Kinetics of Spanish broom peroxidase obeys a Ping-Pong Bi–Bi mechanism with competitive inhibition by substrates

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae

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