Catalase: Bioinformatics Analyses of One of the Key Enzymes in Hydrogen Peroxide Metabolism

6–71RYHPEHU 2019, Brno, Czech Republic

Catalase: Bioinformatics analyses of one of the key enzymes in hydrogen peroxide metabolism

Michaela Kameniarova, Romana Kopecka Department of Molecular Biology and Radiobiology Mendel University in Brno Zemedelska 1, 613 00 Brno CZECH REPUBLIC [email protected]

Abstract: Catalases (CAT) are family of important antioxidant enzymes present in different isoforms and responsible for scavenging of hydrogen peroxide in almost all aerobically living organisms. In plants, they were found both in unicellular and multicellular species. Here, we performed bioinformatics analysis and analysed catalase evolutionary relationship and expression patterns. By comparing expression profiles of CATs and expression profiles of genes related to abiotic stimuli we found that almost 50% of light signalling genes were co-expressed with CATs. Further, by datamining in available resources and structural modelling we pinpointed candidate amino acid residues responsible for CAT thermostability.

Key Words: catalase, H2O2, phylogenetics, abiotic stress, stability

INTRODUCTION

Plants contain several types of enzymes involved in H2O2 metabolism. These include catalases, ascorbate peroxidases, peroxiredoxins, glutathione/thioredoxin peroxidases, and glutathione S- transferases, but only catalases do not require additional cellular reductants (Mhamdi et al. 2010). Catalases are present almost in all aerobically respiring organisms. Within the cell environment, catalases are localized in all major sites of H2O2 production, mostly at peroxisomes, but they were detected in cytosol, mitochondria, and chloroplasts as well (Sharma and Ahmad 2014). Catalases (CAT, 1.11.1.6) are antioxidant enzymes that catalyse decomposition of hydrogen peroxide to water and molecular oxygen (2H2O2 → 2H2O + O2), an important process in defending cells against oxidative damage caused by reactive oxygen species (ROS; Alfonso-Prieto et al. 2009). The H2O2 affects a plant in the concentration-dependent manner. High amounts of H2O2 cause cell damage, potentially leading to cell death, whereas low concentrations can act as a signal regulating speciﬁc biological processes (Zámocký et al. 2012). For the survival and normal development of the plant it is necessary to maintain balance between H2O2 production and its decomposition. Genes for catalases in plants usually create small gene family. Catalase gene family in model plant Arabidopsis consists of three genes encoding CAT1, CAT2 and CAT3. These CAT isoforms show different tissue localization. CAT1 is localized mostly in pollen and seeds, while CAT2 is expressed in photosynthetic tissues and in roots, and CAT3 in vascular tissues (Buzduga et al. 2018). Besides the different localization, they also have a different expression pattern, and the transcription levels depend on time, place and environmental stimuli. Expression studies of CAT2 and CAT3 revealed that they are regulated by circadian rhythm, with CAT2 expression peaking at the beginning of the light period and CAT3 in the dark (Mhamdi et al. 2012). Environmental stresses, such as drought, heat or cold, generally enhance the transcription of CATs, and thus increase catalases’ activity, maintaining redox balance in the cell. Mutation in CAT genes disturbs this redox state. In Arabidopsis, a loss-of-function mutant cat2 reduced catalase activity in leaves by 90% in comparison to Col-0 wild-type plants, but the deletion of CAT1 and CAT3 had only a mild effect on the total catalase activity (Mhamdi et al. 2010, Su et al. 2018).

425 6–71RYHPEHU 2019, Brno, Czech Republic

MATERIAL AND METHODS Selection of organisms and construction of phylogenetic tree The list of organisms and accession numbers of their CAT nucleotide sequences used for phylogenetic reconstruction are summarized in Table 1. The sequences were obtained from ENA (European Nucleotide Archive) and GenBank databases. Multiple sequence alignment and phylogenetic analysis was constructed using the GenomeNet ETE 3 v3.0.0b32 (https://www.genome.jp/tools/ete/; Huerta-Cepas et al. 2016) with the following parameters: Aligner – mafft, Alignment cleaner – none, model tester – none, tree builder fasttree (Price et al. 2009). The constructed tree file was visualized using iTOL v3.0 (http://itol2.embl.de/index.shtml; Letunic and Bork 2006, Letunic and Bork 2011). Table 1 Overview of model organisms and corresponding CATs used in the analysis Organism Accession numbers Arabidopsis thaliana NC_003070.9, NC_003070.9, NC_003075.7 Chlamydomonas reinhardtii NW_001843734.1 Malus domestica ENA|RXH96794|RXH96794.1, ENA|RXH96798|RXH96798.1 Marchantia polymorpha ENA|OAE35245|OAE35245.1, ENA|OAE31579|OAE31579.1 ENA|OAE35247|OAE35247.1 Medicago truncatula NW_013656164.1, NC_016409.2, ENA|KEH36313|KEH36313.1 Nicotiana benthamiana ENA|ACL27272|ACL27272.1, ENA|AFU48609|AFU48609.1 Oryza sativa NC_029257.1, NC_029258.1, NC_029261.1 Plasmodiophora brassicaceae ENA|CEO97147|CEO97147.1 Physcomitrella patens ENA|EDQ83048|EDQ83048.1 Populus trichocarpa ENA|PNT38671|PNT38671.1, NC_037289.1, NC_037286.1 Solanum tuberosum ENA|AAA80650|AAA80650.1, ENA|CAA85470|CAA85470.1 Zea mays ENA|CAA31056|CAA31056.1, ENA|CAA38588|CAA38588.1 ENA|AAC37357|AAC37357.1 Expression patterns of CAT genes To describe the correlation of CAT expression profiles with expression profiles of abiotic-stress related genes, light signalling genes and phytohormones metabolism genes, we searched data from Černý et al. (2018) against the ThaleMine (https://www.araport.org/). Searching for natural CAT mutants In order to obtain information about CAT polymorphism in different natural accessions of Arabidopsis we used an online tool Polymorph 1001 https://tools.1001genomes.org/polymorph/. Prediction of hotspot residues from CAT protein sequences Hotspots in CAT protein sequences of Zea mays were identified by automated multi-step calculation using online tool HotSpot Wizard 3.0. (http://loschmidt.chemi.muni.cz/hotspotwizard). CATs protein sequences were obtained from UniProt and the tertiary structure of CATs was retrieved from repositories of homology models. We performed multiple alignment of selected CAT sequences, calculated hotspots with amino acid residues in which the CAT isoforms differ and modelled the mutation of CAT in these residues to determine its effect on the stability of the individual catalase isoforms. The data about temperature stability of CATs were obtained from enzyme database Brenda (https://www.brenda-enzymes.org/index.php).

RESULTS AND DISCUSSION Evolutionary relationships of CATs in different plant species Catalases are produced by almost all aerobically living organisms ranging from unicellular to multicellular species and their evolution relates to development of the aerobic biosphere on Earth. They are present in multiple molecular forms in plants, creating small gene families. In higher plants,

426 6–71RYHPEHU 2019, Brno, Czech Republic they usually consist of 3 members with varying levels of sequence similarity (Sharma and Ahmad 2014). Phylogenetic reconstruction using maximum likelihood method to the catalase sequences of 11 different organisms resulted in a rooted tree shown in Figure 1. Catalases of the same gene family are often clustered together, while CAT isoforms of some species (Oryza sativa, Zea mays, Populus trichocarpa) are situated on the distinct branches of the tree. CAT of single-cell Chlamydomonas reinhardtii shows the highest evolutionary distance from the rest of the sequences, creating a separated branch. The Arabidopsis genome encodes three CAT isoforms consisting of 492 amino acids (Mhamdi et al. 2010). They share high sequence similarity grouping Arabidopsis CATs together. Figure 1 Rooted phylogenetic tree based on the nucleotide sequences of CAT genes

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Relation of CATs expression patterns with expression patterns of abiotic stress-related genes and phytohormone metabolism genes Catalase is a well-known enzyme involved in biotic and abiotic stress responses. Here, to underscore its role in a stress response, we employed a dataset containing previously reported marker genes in Arabidopsis and compared the expression patterns to that of catalase. The dataset contained genes related to nutrient stress (142 genes), temperature stress (43 genes), drought stress (13 genes), light signalling (27) and hormone metabolism genes (101). Surprisingly, we did not find a significant overlap between Arabidopsis CATs expression and the expression of temperature or drought-stress- related genes. However, we found that almost 50% of analysed light signalling genes and a significant portion of phytohormones metabolism genes were co-expressed with CATs. High level of similarity of CATs expression patterns and light signalling is likely the result of photosynthetic activity and ROS scavenging (Buzduga et al. 2018). In accordance, the light period plays role in regulation of transcript abundance in two of three catalases, CAT2 and CAT3. Their expression level is controlled by day-night rhythm, where the photorespiratory catalase CAT2 shows a peak early in the light period and the CAT3 in dark (Mhamdi et al. 2012). Figure 2 Percentage of abiotic stress markers genes, light signalling genes and phytohormones metabolism genes co-expressed with CATs in Arabidopsis

Temperature stability of CATs and hotspots in CAT protein sequences of Zea mays Hotspot residues are important in the stability of protein-protein interactions, and they always have some specific functions in protein. Thermostability of enzymes is critical for their applicability in biotechnologies. Because the data about temperature stability of CATs in Arabidopsis thaliana are not available, we analysed thermostability of CATs in important crop, Zea mays, wherein the names of catalases corresponds to catalases in Arabidopsis. The Brenda database showed that the most thermo stable isoform of CAT in Zea mays is CAT2, which is stable at 45 °C, while the half-life of CAT1 is 12.5 minutes and half-life of CAT3 is 18 minutes (Chandlee et al. 1983). By designing specific mutations, we identified amino acid residues that could be responsible for thermostability or thermolability of different CAT isoforms in Zea mays and they are shown in Figure 3. We found that mutation of CAT1 Met 185 to Thr, normally present in CAT3, leads to its destabilization, but on the concurrent mutation of Thr 343 to Cys has a positive effect on CAT3 stability. In CAT2, mutation of Val 288 to Thr and mutation of Thr 342 to Cys, normally present in CAT3 amino acid sequence, has a destabilizing effect on CAT3, and this could be a reason why CAT3 is not as thermo stable as CAT2. Arabidopsis thaliana ecotypes with mutation in CAT Catalase is a well-optimized enzyme with one of the highest known turnover rates (Černý et al. 2018). However, its mutation does not have to be lethal in optimal growth conditions. Here,

428 6–71RYHPEHU 2019, Brno, Czech Republic to investigate natural mutations rates, we employed the 1001 Arabidopsis genome database. Surprisingly, we found only four Arabidopsis thaliana accessions carrying a significant mutation in the CAT gene, and all these were mutants in CAT3. The further analysis showed that these mutants IP-Mdd- 0, IP-Esn-2, IP-Moe-0 and IP-Tau-0 originated from the same area in Spain. This finding indicates that CAT2 and CAT1 are highly conserved and only CAT3 seems to be prone to mutation. This could correlate with the reports showing that knock-out of CAT3 does not have a significant impact on catalase activity and the plants can grow normally (Mhamdi et al. 2010). Figure 3 Protein structure of Zea mays CAT isoforms. Amino acid residues potentially responsible for thermostability/thermolability of different CAT isoforms are visualised by yellow colour. The localization of their catalytic pockets is shown by orange colour.

CONCLUSION

The main role of catalases in organisms is decomposition of H2O2, maintaining redox homeostasis in cells. We found that catalases in plants share expression pattern with almost 50% of light signalling genes and with significant portion of phytohormones metabolism genes. In our study we also demonstrated evolutionary relationship of catalases in different plant species and we identified four ecotypes of Arabidopsis thaliana having unfunctional gene for CAT3. At the end we analysed amino acid residues responsible for temperature stability of Zea mays CAT isoforms, where we found that mutation of CAT2 Val 288 to Thr and mutation of Thr 342 to Cys, which are present in CAT3 amino acid sequences, cause destabilization of enzyme, what could be the reason for lower temperature stability of CAT3.

ACKNOWLEDGEMENTS The research was financially supported by AF-IGA-IP-2019/IP081 (Internal Grant Agency of Faculty of AgriSciences, Mendel University in Brno). The authors would like to express their thanks to Markéta Luklová and Martin Černý for support and constructive comments.

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