Enzyme Research Group
Current Group Members
Jakub Drożak, PhD, DSc (Principal Investigator)
Apolonia Witecka, MSc (PhD student, UW)
Klaudia Ślusarczyk, MSc (PhD student, IMID)
Julia Kamińska, MSc (PhD student, UW)
Paulina Emmel, BSc (MSc student)
Mikołaj Witczak (BSc student)
Aims and Objectives
In our studies, we are mainly interested in the molecular identification of vertebrate orphan enzymes followed by a detailed molecular and biochemical characterization of these proteins. Orphan enzymes are biological catalysts that have been already detected in biological systems (cells, tissues or organs), but their genes, the molecular structures and the biochemical properties remain unknown. For these reasons, very little is known on their physiological role, the regulation of their enzymatic activity, and finally, their importance in health and disease.
Collaborating with prof. Agnieszka Rygiel (Institute of Mother and Child, Warsaw), we also analyze the functional consequences of novel mutations in human genes identified in Polish patients, focusing on their impact on enzyme activity.
Methods
Biochemistry
- chromatographic, radiochemical and spectrophotometric assays of enzyme activity,
- multi-step purification of native and recombinant proteins, employing various chromatographic methods (e.g. ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, affinity chromatography),
- analysis of protein purity (SDS-PAGE, Western-blotting)
- identification of proteins and their post-translational modifications with the use of hybrid mass spectrometry (Q-TOF),
- molecular identification of low molecular weight products of enzyme activity (HPLC-MS, IC-MS).
Molecular Biology
- molecular cloning of the open-reading frames coding for prokaryotic and eukaryotic proteins of our interest,
- site-directed mutagenesis of proteins,
- genome editing in mammalian cell lines using CRISPR/Cas9 technique,
- small- to medium-scale production of recombinant proteins in E. coli or in mammalian cell lines,
- purification of recombinant proteins, employing FPLC technique.
Main Research Findings
- identification of the HSD17B14 enzyme as the mammalian L-fucose dehydrogenase that initializes L-fucose breakdown in the liver and kidneys, and its biochemical characterization,
- identification of the BDH2 enzyme as vertebrate 4-oxo-L-proline reductase and its biochemical and functional characterization,
- identification of the SETD3 protein as a long-sought actin-specific histidine N-methyltransferase in animals; biochemical and functional characterization of this enzyme,
- identification of mechanisms determining the substrate specificity of SETD3,
- identification of vertebrate HNMT-like and C9orf41 genes as ones that encode carnosine N-methyltransferase – the terminal enzyme in anserine (β-alanyl-Nπ-methyl-L-histidine) biosynthesis pathway,
- identification of ACSF4-U26 protein as a β-alanine-activating enzyme which is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.
Alumni (PhD theses)
Sebastian Kwiatkowski (2023) The characterization of the novel enzymatic activities of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2)
Alumni (MSc theses)
Julia Kamińska (2024) Functional analysis of selected variants of the human GTP cyclohydrolase 1 in the context of Segawa’s syndrome development
Varvara Kazak (2024) Preliminary biochemical characterization of rat L-fucose dehydrogenase (EC 1.1.1.122)
Kacper Domżał (2023) Preliminary functional analysis of a selected point mutation in the gene of human GTP cyclohydrolase I (GHC1) in the context of Segawa’s disease.
Natalia Głogowska (2022) Preliminary functional analysis of selected point mutations in the gene of human aromatic L-amino acid decarboxylase (AADC) in the context of inborn deficiency of this enzyme.
Michał Zaród (2020) Functional analysis of selected point mutations in gene encoding human calcium channel TRPV6 associated with development of chronic pancreatitis in children
Weronika Tomaka (2020) Purification and preliminary molecular identification of rat S100A9 protein methyltransferase.
Maria Bożko (2018) Purification and preliminary molecular identification of rat 4-oxo-L-proline reductase (EC 1.1.1.104)
Agnieszka Seliga (2017) Preliminary biochemical characterization of human β-actin histidine N-methyltransferase (EC 2.1.1.85)
Sebastian Kwiatkowski (2016) Purification and molecular identification of rat skeletal muscle β-actin histidine N-methyltransferase
Maria Piecuch (2014) Biochemical characterization of UPF0586 proteins human C9orf41 homologes.
Olga Poleszak (2013) Purification and molecular identification of rat (Rattus norvegicus) carnosine N-methyltransferase (EC 2.1.1.22)
Łukasz Chrobok (2013) Molecular identification of chicken Carnosine N-methyltransferase (EC 2.1.1.22).
Beata Kądziołka (2012) Revealing some secrets… An attempt to identify the reaction catalyzed by Acyl-CoA Synthetase Family Member 4
Alumni (BSc theses)
Paulina Emmel (2023) Inborn errors of serotonin and catecholamine neurotransmitters biosynthesis in human beings
Emilia Staszór (2022) Preliminary investigation on enzymatic activity of the two domain methyltransferase from Calditerrivibrio nitroreducens
Joint supervision by Rafał Augustyniak (Faculty of Chemistry) and Jakub Drożak (Faculty of Biology)
Julia Kamińska (2022) L-fucose – metabolism and biological importance in mammals.
Varvara Kazak (2021) Enzymes as the target for antiviral drugs.
Kamil Szostak (2019) A brief characterization of ATP-grasp enzymes.
Kacper Domżał (2019) The use of enzymes to detoxify chemical warfare agents.
Michał Zaród (2018) Biological Significance of Protein Methylation.
Weronika Tomaka (2017) Enzyme replacement therapy in inborn errors of metabolism
Maria Bożko (2016) Enzyme Replacement Therapy during pancreatic cancer
Izabela Niemyjska (2015) Carnosine and related dipeptides: physiological role and prospects of use in the treatment of human diseases
Sebastian Kwiatkowski (2014) Enzymes of metabolite proofreading – physiological importance
Łukasz Chrobok (2011) Putative imidazoline receptors as a target for drugs action
Joanna Sekuła (2011) Nonribosomal peptide synthetases: mechanism of synthesis and pharmacological importance of products
Joanna Kowalewska (2007) Rasagiline- a new drug in the treatment of Parkinson’s disease.
Selected Papers
Maas MN, Bilgin N, Moesgaard L, Hintzen JCJ, Drozak A, Drozak J, Kongsted J, Mecinović J. (2024) Examining prestructured β-actin peptides as substrates of histidine methyltransferase SETD3. Sci Rep. 14(1):26439.
https://www.nature.com/articles/s41598-024-76562-z.pdf
Witecka A, Kazak V, Kwiatkowski S, Kiersztan A, Jagielski AK, Kozminski W, Augustyniak R, Drozak J. (2024) Hydroxysteroid 17-β dehydrogenase 14 (HSD17B14) is an L-fucose dehydrogenase, the initial enzyme of the L-fucose degradation pathway. J Biol Chem. 300(8):107501.
https://www.jbc.org/action/showPdf?pii=S0021-9258%2824%2902002-7
Akagashi M, Watanabe S, Kwiatkowski S, Drozak J, Terawaki SI, Watanabe Y. (2024) Crystal structure of L-2-keto-3-deoxyfuconate 4-dehydrogenase reveals a unique binding mode as a α-furanosyl hemiketal of substrates. Sci Rep. 14(1):14602.
https://www.nature.com/articles/s41598-024-65627-8.pdf
Al-Fakhar MSQ, Bilgin N, Moesgaard L, Witecka A, Drozak J, Kongsted J, Mecinović J. (2023) The Role of Trp79 in β-Actin on Histidine Methyltransferase SETD3 Catalysis. Chembiochem. 24(21):e202300490. https://doi.org/10.1002/cbic.202300490
Bisello G, Kusmierska K, Verbeek MM, Sykut-Cegielska J, Willemsen MAAP, Wevers RA, Szymańska K, Poznanski J, Drozak J, Wertheim-Tysarowska K, Rygiel AM, Bertoldi M. (2022). The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role. Cell Mol Life Sci. 2022; 79(6):305. https://link.springer.com/article/10.1007/s00018-022-04343-w
Hintzen, J., Ma, H., Deng, H., Witecka, A., Andersen, S. B., Drozak, J., Guo, H., Qian, P., Li, H., & Mecinović, J. (2022). Histidine methyltransferase SETD3 methylates structurally diverse histidine mimics in actin. Protein Science: a publication of the Protein Society, 31(5), e4305. https://doi.org/10.1002/pro.4305
Kwiatkowski, S., Bozko, M., Zarod, M., Witecka, A., Kocdemir, K., Jagielski, A. K., & Drozak, J. (2022). Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104). The Journal of Biological Chemistry, 298(3), 101708. https://doi.org/10.1016/j.jbc.2022.101708
Bilgin, N., Moesgaard, L., Maas, M. N., Hintzen, J., Witecka, A., Drozak, J., Kongsted, J., & Mecinović, J. (2022). Importance of Ile71 in β-actin on histidine methyltransferase SETD3 catalysis. Organic & Biomolecular Chemistry, 20(8), 1723–1730. https://doi.org/10.1039/d1ob02430b
Witecka, A., Kwiatkowski, S., Ishikawa, T., & Drozak, J. (2021). The Structure, Activity, and Function of the SETD3 Protein Histidine Methyltransferase. Life (Basel, Switzerland), 11(10), 1040. https://doi.org/10.3390/life11101040
Oracz, G., Zaród, M., Ewers, M., Laumen, H., Gambin, T., Kamiński, P., Grabowska, I., Drożak, A., Kwiatkowski, S., Wertheim-Tysarowska, K., Kołodziejczyk, E., Domaszewicz, A., Dorożko, B., Kosińska, J., Głuszek, S., Kozieł, D., Płoski, R., Rosendahl, J., Witt, H., Drożak, J., Rygiel, A. M. (2021). Loss of function TRPV6 variants are associated with chronic pancreatitis in nonalcoholic early-onset Polish and German patients. Pancreatology : Official Journal of the International Association of Pancreatology (IAP), 21(8), 1434–1442. https://doi.org/10.1016/j.pan.2021.09.005
Kwiatkowski S, Drozak J. Protein Histidine Methylation. Curr Protein Pept Sci. 2020;21(7):675-689.https://www.eurekaselect.com/180353/article
Guo Q, Liao S, Kwiatkowski S, Tomaka W, Yu H, Wu G, Tu X, Min J, Drozak J, Xu C. Structural insights into SETD3-mediated histidine methylation on β-actin. Elife. 2019; 8:e43676. https://elifesciences.org/articles/43676
Kwiatkowski S, Seliga AK, Vertommen D, Terreri M, Ishikawa T, Grabowska I, Tiebe M, Teleman AA, Jagielski AK, Veiga-da-Cunha M, Drozak J. SETD3 protein is the actin-specific histidine N-methyltransferase. Elife. 2018; 7. pii: e37921. https://elifesciences.org/articles/37921
Kwiatkowski S, Kiersztan A, Drozak J. Biosynthesis of carnosine and related dipeptides in vertebrates. Curr Protein Pept Sci. 2018;19(8):771-789. http://www.eurekaselect.com/160120/article
Drozak J, Piecuch M, Poleszak O, Kozlowski P, Chrobok L, Baelde HJ, de Heer E. UPF0586 Protein C9orf41 Homolog Is Anserine-producing Methyltransferase. J Biol Chem. 2015; 290(28):17190-205. http://www.jbc.org/content/290/28/17190.long
Drozak J, Veiga-da-Cunha M, Kadziolka B, Van Schaftingen E. Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase. FEBS J. 2014 Mar;281(6):1585-97. https://doi.org/10.1111/febs.12725
Drozak J, Chrobok L, Poleszak O, Jagielski AK, Derlacz R. Molecular identification of carnosine N-methyltransferase as chicken histamine N-methyltransferase-like protein (hnmt-like). PLoS One. 2013 May 21;8(5):e64805. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064805
Research Grants
(Principal Investigator –Jakub Drożak)
“Biochemical and functional characterization of mammalian 2-keto-3-deoxy-L-fuconate dehydrogenase” – The National Science Centre (Poland) grant, Opus program (26th edition), 2024-2026.
„Biochemical characterization of human SETD3 methyltransferase” – The National Science Centre (Poland) grant, Opus program (14th edition), 2018-2023.
„Molecular and biochemical characterization of eukaryotic C9orf41 protein.” – The National Science Centre (Poland) grant, Opus program (6th edition), 2014-2016.
„Molecular and biochemical characterization of Acyl-CoA Synthase family member 4, a mysterious enzyme of mammalians” – The Foundation for Polish Science grant, Homing plus program, 2011-2013.
„Molecular and biochemical characterization of carnosine N-methyltransferase (EC 2.1.1.22)” – The Ministry of Science and Higher Education (Poland) grant, Iuventus Plus program, 2010-2011.
FUNDING AGENCIES:
