Investigating the active centre of the Scytalidium thermophilum catalase

Date

2013-04-01

Author

YÜZÜGÜLLÜ, YONCA
Trinh, Chi H.
Fairhurst, Lucy
Ögel, Zümrüt Begüm
McPherson, Michael J.
Pearson, Arwen R.

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

160
views

0
downloads

Almost all monofunctional haem catalases contain a highly conserved core containing the active site, which is connected to the exterior of the enzyme by three channels. These channels have been identified as potential routes for substrate flow and product release. To further investigate the role of these molecular channels, a series of mutants of Scytalidium thermophilum catalase were generated. The three-dimensional structures of four catalase variants, N155A, V123A, V123C and V123T, have been determined at resolutions of 2.25, 1.93, 1.9 and 1.7 angstrom, respectively. The V123C variant contains a new covalent bond between the S atom of Cys123 and the imidazole ring of the essential His82. This variant enzyme has only residual catalase activity and contains haem b instead of the normal haem d. The H82A variant demonstrates low catalase and phenol oxidase activities (0.2 and 20% of those of recombinant wild-type catalase-phenol oxidase, respectively). The N155A and N155H variants exhibit 4.5 and 3% of the wild-type catalase activity and contain haem d, showing that Asn155 is essential for catalysis but is not required for the conversion of haem b to haem d. Structural analysis suggests that the cause of the effect of these mutations on catalysis is the disruption of the ability of dioxygen substrates to efficiently access the active site. Additional mutants have been characterized biochemically to further probe the roles of the different channels. Introducing smaller or polar side chains in place of Val123 reduces the catalase activity. The F160V, F161V and F168V mutants show a marked decrease in catalase activity but have a much lower effect on the phenol oxidase activity, despite containing substoichiometric amounts of haem.

Subject Keywords

Biophysics, Genetics, Biochemistry, Structural Biology, Condensed Matter Physics

URI

https://hdl.handle.net/11511/52445

Journal

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS

DOI

https://doi.org/10.1107/s1744309113004211

Collections

Graduate School of Natural and Applied Sciences, Article

Suggestions

OpenMETU
Core

Investigating the malleability of RNA aptamers İlgü, Müslüm; Lamm, Monica H.; Nilsen-Hamilton, Marit (Elsevier BV, 2013-09-15) Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics. Here we discuss the integration of biochemic...
The low spin - high spin equilibrium in the S-2-state of the water oxidizing enzyme Boussac, Alain; Ugur, Ilke; Marıon, Antoıne; Sugiura, Miwa; Kaila, Ville R. I.; Rutherford, A. William (Elsevier BV, 2018-05-01) In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S-0 to S-4) before water is oxidized and O-2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S-2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S-2(LS) and S-2(HS) is pH depend...
Investigation of the cellular mechanisims underlying the Carboxypeptidase E mutation Kaşıkçı, Feride; Yanık, Tülin; Department of Biology (2014) Carboxypeptidase E (CPE) is an enzyme expressed in both endocrine and neuroendocrine cells functioning as both an exopeptidase and a sorting receptor. Recently, it has been reported that CPE plays a role in preventing neuronal cell death in the CA3 hippocampus so as to maintain normal cognitive function in the adult brain. Studies on CPE-knockout mice showed total degeneration of neurons in the CA3 region of the hippocampus in adult mice 4 weeks of age and older. Additionally, increased CPE expression was i...
The effect of valine substitution for glycine in the dimer interface of citrate synthase from Thermoplasma acidophilum on stability and activity Kocabıyık, Semra (Elsevier BV, 2000-08-28) To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (K-m for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., V-max, K-cat and...
The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum Kocabıyık, Semra; Russel, RJM; Danson, MJ; Hough, DW (Elsevier BV, 1996-07-05) In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate s...

Citation Formats

Y. YÜZÜGÜLLÜ, C. H. Trinh, L. Fairhurst, Z. B. Ögel, M. J. McPherson, and A. R. Pearson, “Investigating the active centre of the Scytalidium thermophilum catalase,” ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, pp. 369–375, 2013, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/52445.