Chair of General and Microbial Biochemistry
Head of the Chair: prof. Juhan Sedman
Contact details: Riia23b -218, Phone +372 7375837, juhan.sedman [ät] ut.ee
The yeast group
Leader prof. Juhan Sedman
Contact: Estonia, Tartu, Riia 23b-218, Tel: 737 5837, juhan.sedman [ät] ut.ee
• Mitokochondrial DNA replication and recobination
• Mitochondrial DNA helicases
Juhan Sedman, PhD, juhan.sedman [ät] ut.ee
Tiina Sedman, researcher, PhD, tiina.sedman [ät] ut.ee
Sirelin Sillamaa, PhD student
Vlad-Julian Piljukov, PhD student
Natalja Garber, PhD student (on academic leave)
Mitochondria are organelles that have their own genomic DNA. We are interested in the mechanisms by which the mitochondrial genome is propagated during the cell cycle. We try to understand what changes in the structure of DNA molecules take place during synthesis and how mitochondrial DNA replication and recombination are related. We are also interested in enzymes involved in mitochondrial DNA metabolism - DNA polymerase, which synthesizes mitochondrial DNA; nucleases required for DNA stability and helicases, which are motor enzymes that modulate the structure of nucleic acids.
Analysis of mitochondrial DNA topology and stable inheritance
Analysis by our group has shown that mitochondrial DNA forms complex higher-order network structures in yeast and cannot be described by the simple ring structures depicted in the textbooks. Our data indicate that such DNA structures are likely to result from recombination and that yeast mitochondria do not have the specific DNA synthesis initiation structures that should be present when using RNA primers.
The S. cerevisiae model organism allows us to systematically analyze the effects of various enzymes on mitochondrial DNA stability, as baker's yeast can grow without a functional respiratory chain. We have constructed yeast strains in which RNA synthesis in mitochondria is eliminated, allowing the identification of factors important in recombinant DNA synthesis.
Function of mitochondrial DNA helicases
As a rule, the DNA helicase is involved in DNA replication in various systems, and its task is to unravel the strands at the replication fork. As a result of our laboratory work, two DNA helicases have been identified in the mitochondria, but it has also been found that the classical replicative helicase in yeast mitochondria is apparently absent. For functional analysis of mitochondrial DNA helicases, we use a combination of biochemical experiments with purified proteins and in vivo analysis. Thus, we have shown that these mirochondrial helicases have specificity for branched-chain DNA molecules, suggesting their role in recombinant processes.
1. Piljukov VJ, Garber N, Sedman T, Sedman J.(2020) Irc3 is a monomeric DNA branch point-binding helicase in mitochondria of the yeast Saccharomyces cerevisiae. FEBS Lett. 594(19):3142-3155.
2. Sedman T, Garber N, Gaidutšik I, Sillamaa S, Paats J, Piljukov VJ, Sedman J. (2017) Mitochondrial helicase Irc3 translocates along double-stranded DNA. FEBS Lett. 591(23):3831-3841.
3. Wanrooij PH, Engqvist MKM, Forslund JME, Navarrete C, Nilsson AK, Sedman J, Wanrooij S, Clausen AR, Chabes (2017) Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired. Proc Natl Acad Sci U S A. ;114(47):12466-12471.
4. Gaidutšik I, Sedman T, Sillamaa S, Sedman J (2016) Irc3 is a mitochondrial DNA branch migration enzyme. Sci Rep. 6:26414.
5. Sedman T, Gaidutšik I, Villemson K, Hou Y, Sedman J. (2014) Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance. Nucleic Acids Res. 42(21):13214-27.
6. Gerhold JM, Sedman T, Visacka K, Slezakova J, Tomaska L, Nosek J, Sedman J. (2014) Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.J Biol Chem. 289(33):22659-70.
7. Aun A, Tamm T, Sedman J. (2012) Dysfunctional mitochondria modulate cAMP-PKA signaling and filamentous and invasive growth of Saccharomyces cerevisiae. Genetics. 2013 : 467-81.
8. Reimand J, Aun A, Vilo J, Vaquerizas JM, Sedman J, Luscombe NM.(2012) m:Explorer: multinomial regression models reveal positive and negative regulators of longevity in yeast quiescence. Genome Biol. 13(6):R55
9. Viikov K, Jasnovidova O, Tamm T, Sedman J. (2012) C-terminal extension of the yeast mitochondrial DNA polymerase determines the balance between synthesis and degradation. PLoS One. 2012;7(3):e33482.
10. Gerhold JM, Aun A, Sedman T, Jõers P, Sedman J. (2010) Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.. Mol.Cell. 2010:851-61
11. Lõoke M, Reimand J, Sedman T, Sedman J, Järvinen L, Värv S, Peil K, Kristjuhan K, Vilo J, Kristjuhan A. (2010) Relicensing of transcriptionally inactivated replication origins in budding yeast. J. Biol Chem. 285(51):40004-11.
12. Viikov K, Väljamäe P, Sedman J. (2011) Yeast mitochondrial DNA polymerase is a highly processive single-subunit enzyme. Mitochondrion. : 20;119-26.
Cellulose research group
Head of the group: Associate professor Priit Väljamäe
Contact: Riia 23b – 202, Tel. 737 5823, priit.valjamae [ät] ut.ee
• Enzymatic degradation of recalcitrant polysaccharides (cellulose and chitin)
• Redox reactions in lignocellulose degradation
Priit Väljamäe, associate professor, PhD, priit.valjamae [ät] ut.ee
Silja Kuusk, researcher, PhD, silja.kuusk [ät] ut.ee
Jürgen Jalak, researcher, PhD, jyrgen.jalak [ät] ut.ee
Alexander Rannar, master student
Alexey Nesterovich, bachelor student
Alisa Kamnerov, bachelor student
- Structural polysaccharides – cellulose and chitin – are the most abundant polysaccharides on the Earth and represent a huge reservoir of renewable carbon. Their enzymes aided valorization provides green and sustainable alternative to traditional petroleum-based industry. However, the crystalline structure of cellulose and chitin makes them recalcitrant towards enzymatic degradation. Development of optimal enzyme mixtures assumes in depth understanding of the mode of action and kinetics of individual enzyme components. Hence, detailed analysis of enzyme kinetics and mechanism is on the main focus of our research group.
- Enzymatic degradation of recalcitrant polysaccharides (cellulose and chitin)
Owing to their crystalline structure, the enzymatic degradation of cellulose and chitin takes place on the solid liquid interface. This is a complex heterogeneous catalysis that involves number of intermediate steps. Identification of the rate limiting step (bottleneck) is prerequisite for accelerating the whole reaction. The key approach of our research is the development of novel methods that enable to measure the rates of individual intermediate steps of complex catalysis. In this way we have measured processivity and the values of rate constants of dissociation, association and glycosidic bond hydrolysis by glycoside hydrolases. Besides traditional glycoside hydrolases we are also focused on lytic polysaccharide monooxygenases (LPMOs). LPMOs are recently discovered redox enzymes that catalyze oxidative degradation of recalcitrant polysaccharides. Understanding the mechanism and kinetics of LPMO catalysis may help better harnessing of their catalytic potential and increase the efficiency of degradation and modification of recalcitrant polysaccharides.
- Redox reactions in lignocellulose degradation
LPMO catalysis needs electrons (reductant) as well as H2O2/O2 co-substrate (oxidant). Recognition of the importance of LPMOs has also boosted the research of the redox reactions that contribute to degradation of lignocellulose (the main component of plant cell walls). Here we focus on the development of enzyme cascades that enable to establish optimal conditions for LPMO activity and stability in the complex redox active environment. Besides LPMOs we are interested in different enzymes like laccases, peroxidases and oxidases that are active on lignin or its degradation products.
1. Kont, R., Bissaro, B., Eijsink, V. G. H., and Väljamäe, P. (2020) Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs). Nat. Commun. 11:5786
2. Vermaas, J. V., Kont, R., Beckham, G. T., Crowley, M. F., Gudmundsson, M., Sandgren, M., Ståhlberg, J., Väljamäe, P., and Knott, B. C. (2019) The dissociation mechanism of processive cellulase. Proc. Natl. Acad. Sci. USA. 116, 23061-23067
3. Kont, R., Pihlajaniemi, V., Borisova, A. S., Aro, N., Marjamaa, K., Loogen, J., Büchs, J., Eijsink, V. G. H., Kruus, K., and Väljamäe, P. (2019) The liquid fraction from hydrothermal pretreatment of wheat straw provides lytic polysaccharide monooxygenases with both electrons and H2O2 co-substrate. Biotechnol. Biofuels 12:235
4. Kuusk, S., Kont, R., Kuusk, P., Heering, A., Sørlie, M., Bissaro, B., Eijsink, V. G. H., and Väljamäe, P. (2019) Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase. J. Biol. Chem. 294, 1516-1528
5. Kuusk, S., Bissaro, B., Kuusk, P., Forsberg, Z., Eijsink, V. G. H., Sørlie, M., and Väljamäe, P. (2018) Kinetics of H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase. J. Biol. Chem. 293, 523-531
6. Kuusk, S., and Väljamäe, P. (2017) When substrate inhibits and inhibitor activates: implications of β-glucosidases. Biotechnol. Biofuels 10:7
7. Kurašin, M., Kuusk, S., Kuusk, P., Sørlie, M., and Väljamäe, P. (2015) Slow off-rates and strong product binding are required for processivity and efficient degradation of recalcitrant chitin by family 18 chitinases. J. Biol. Chem. 290, 29074-29085
8. Kuusk, S., Sørlie, M., and Väljamäe, P. (2015) The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes. J. Biol. Chem. 290, 11678-11691
9. Jalak, J., Kurašin, M., Teugjas, H., Väljamäe, P. (2012) Endo-exo synergism in cellulose hydrolysis revisited. J. Biol. Chem. 287, 28802-28815.
10. Kurašin, M., Väljamäe, P. (2011) Processivity of cellobiohydrolases is limited by the substrate. J. Biol. Chem. 286, 169 – 177.
Research group of Drosophila mitochondria
Head of the group: Research Fellow Priit Jõers
Contact details: Phone +372 7375037, priit.joers [ät] ut.ee
1. Relationships between mitochondrial DNA synthesis and transcription
2. The effect of mitochondrial DNA stability on the overall metabolic balance
3. The effect of environmental and induced stress conditions on metabolic balance of animals.
1. Mitochondrion is best known as the major site of ATP synthesis, but it is also involved in many other metabolic processes, e.g. synthesizing a number of metabolites that are important for various cellular processes. Vast majority of proteins that carry out these functions are transported from cytosol, since only a small part (13 proteins) of the mitochondrial proteome is synthesized within this organelle. However, these 13 proteins are vital parts of electron transport chain (ETC) complexes. This system creates a proton gradient for ATP synthesis, also generating a mitochondrial membrane potential which is required for the transport of proteins into the mitochondria. These 13 proteins are encoded by mitochondrial DNA (mtDNA), a remnant of the genome of a mitochondrial free-living ancestor. Therefore all mitochondrial processes depend on the normal maintenance of mtDNA. Disorders in integrity and/or function of mtDNA cause severe, often fatal, pathologies in humans. The topic of my research is to find out how mtDNA synthesis and transcription are coordinated and to what extent are these two processes intertwined. As a result of my work, I have discovered that a tight regulation of DNA and RNA synthesis is necessary to avoid their collisions on the mtDNA template. When this occurs, both processes are disrupted, with severe downstream effects on survival and metabolism in the Drosophila model organism. Moreover, transcription also supports DNA synthesis, as replication can only proceed if the RNA strand generated by transcription hybridizes with the lagging DNA strand, providing RNA primers for the synthesis of that strand.
2. Similar to nuclear chromatin, mitochondrial DNA exists in DNA-protein complex termed mitochondrial nucleoid. Many of these proteins are directly involved in DNA synthesis and gene expression - DNA and RNA polymerases, transcription factors, helicase etc. However, numerous enzymes have been identified in nucleoids that play a central role in important metabolic pathways. It is therefore possible that the stability of mtDNA and its in vivo context may also affect metabolism directly, not only through ETC. So far, however, there is only circumstantial evidence of such connections. I have constructed a mechanism based on the bacterial endonuclease EcoBI that is capable of influencing mtDNA in vivo in a Drosophila. As a result, I have discovered a novel link between mtDNA and general metabolism, where disturbances in mtDNA stability lead to an inability to consume carbohydrates and reorient metabolism towards lipid oxidation. This is caused by two phenomena - both glucose transport into the cell and subsequent catabolism by glycolysis are inhibited. These changes are very similar to the processes that cause type I diabetes in humans, therefore my discovery on this role of mtDNA provides novel insight to the onset of this metabolic pathology.
3. In addition to the research topics listed above, I also investigate the effects of stress due to changing environmental conditions on the overall metabolism of Drosophila and other insects. These stress conditions can be either naturally occurring (e.g. anthropogenic changes in the environment) or induced in laboratory conditions. As an example of the latter, I and my collaborators have found that predator stress causes significant changes in both brain-specific and general, systemic metabolism of fruit flies. These results, which have not yet been published, suggest causal relationships between psychological stress conditions and environmental conditions that and changes causing pathologies.