Biochemistry of denitrification bacteria
Distribution of electron flow in branched respiratory pathways. Intracellular nitrate transport. Enzymology of flavin-dependent oxidoreductases. Feedback of bacterial proteome on changes of growth conditions.
prof. RNDr. Igor Kučera, DrSc.
Group leader
Igor Kučera finished his studies in biochemistry in the year 1979 at the Jan Evangelista Purkyně University (now MUNIN) in Brno. In the same year, he defended his RNDr. thesis and after the change of political system in 1989, he was allowed to defend his DrSc. thesis. In 1992 he obtained the position of associate professor based on his habilitation thesis. In 1998 he became a full professor. His teaching involves courses in Enzymology, Bioenergetics, Non-equilibrium Systems and Advanced Biochemistry. He is also a doctoral board chairman and guarantor of the doctoral study programme Biochemistry.
About Igor Kučera
Contact information:
- Office – C05/234
- Phone – 549 49 5392
- E-mail – ikucera@chemi.muni.cz
Education
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1998 – professor of Biochemistry, SCI MU
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1992 – assistant professor of Biochemistry, SCI MU
- Nitric oxide in the denitrification path of P. denitrificans bacterium
- 1992 – DrSc., Czechoslovak academy of sciences
- Regulation of denitrification pathways in P. denitrificans bacterium
- 1979 – RNDr., SCI UJEP (MU)
- Reactions catalyzed by alcoholdegydronase in the presence of two substrates and a coenzyme – analytical application and kinetics
Research area
The Kucera group focuses on investigating biological denitrification (i.e., microbial reduction of nitrate to gaseous nitrogen) using the common soil bacterium Paracoccus denitrificans as a model organism. The main goal is to shed light on how the regulation of electron flow from growth substrates to the terminal respiratory electron acceptors, oxygen and nitrogen oxocompounds, and to the energy storage polymer polyhydroxybutyrate takes place. Gene knockout and overexpression methods together with whole-cell biochemical assays are employed for the examination of functional significance of individual genes. For illustration, it has recently been found that mutational inactivation of some enzymes in the respiratory chain removes the inhibitory effect of oxygen on nitrate reduction. Cells able to denitrify under aerobic conditions could potentially be used for bioremediation of nitrate contaminated water and reduction of emission of the greenhouse gas nitrous oxide.
The second line of research concerns interrogating a group of cytoplasmic flavin mononucleotide dependent enzymes associated with the oxidative stress response of P. denitrificans. The structures of these proteins have been resolved by X-ray crystallography experiments and deposited into the public Protein Data Bank (PDB ID: 3U7R, 4XHY, 4XJ2, 7PLE, 7QW4). Despite substantial structural homology the enzymes differ among themselves in substrate specificity, displaying different reductase activities toward structurally divergent substrates such as benzoquinone derivatives, superoxide, oxygen, and organoarsenic compounds. An effort is continuously underway to determine the reasons for these differences in terms of active site arrangement and participation of individual amino acid residues. Transcription factors involved in regulating transcript levels of these enzymes are also identified and studied.
Recent publications
FerB-related bacterial flavoproteins have a positively charged arginine residue near the bound FMN cofactor. It was previously thought to form a binding site for chromium or uranium oxoanions, which are inorganic substrates undergoing enzyme-catalyzed reduction. The publication shows that the true physiological anionic substrate can be superoxide, a cytotoxic product of one-electron oxygen reduction. The superoxide reductase activity of the enzyme appears to play a role in cells in protecting against oxidative stress.
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Flavoprotein oxidases catalyze the oxidation of substrates by oxygen, which is reduced to hydrogen peroxide. These enzymes generally contain a tightly bound flavin cofactor. The publication describes an enzyme that binds flavin only freely and formally catalyzes the three-substrate reaction FMN (flavin) + NADH (physiological electron donor) + O2 (electron acceptor). The described enzyme may represent an evolutionary intermediate between flavin reductases and flavoprotein oxidases.
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Aerobic respiration is performed at the cellular level by a sequence of redox electron transfer reactions from substrates to oxygen in the respiratory chain. An undesirable side process is the formation of superoxide and other reactive oxygen species (ROS). The publication shows that ROS damage the respiratory chain itself by direct inactivation of input dehydrogenases. Another mechanism, valid for the terminal cbb3-type oxidase is inactivation of the positive-acting transcription factor FnrP.
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The respiratory chain of the studied bacterium contains three different terminal oxidases capable of reducing oxygen. Their physiological role has not yet been elucidated. The publication shows that the terminal oxidase of the cbb3-type significantly increases the competitive success of the bacterium during growth in mixed cultures limited by oxygen and its ability to form a biofilm. Mutational inactivation of the enzyme causes that the initially anaerobic reduction of nitrate to nitrogen (denitrification) can take place even in the presence of oxygen.
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Cell resistance to arsenic compounds is mediated by the ars operon. One of the encoded proteins is ArsH, previously considered an organoAs (III) oxidase. Contrary to this notion, P. denitrificans ArsH was found to catalyze only the reverse reaction - the reduction of organoAs (V). The obtained results suggest that ArsH plays a more general protective role under oxidative stress and not only in arsenic detoxification.
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The publication brings new structural information about the flavin-dependent NADH oxidase described earlier (Sedláček & Kučera, Mol. Microbiool. 2019, 112, 166-183). By combining enzymological approaches (stationary and pre-stationary enzyme kinetics, analysis of the dependence of kinetic parameters on pH, inhibition by modifying agents specific for individual types of amino acid residues) with the determination of the crystal structure and site-directed mutagenesis, it was possible to identify three amino acid residues key for catalysis. His-117 hydrogen bonds to the N3 atom of the isoalloxazine ring of FMN and thereby fixes the ring in the optimal position for the redox reaction with NADH. The interaction of the amino group of Lys-82 with the carboxamide group of NADH prevents the free rotation of the nicotinamide ring around the N1-C1' bond and causes the hydride transfer from NADH to FMN to proceed stereospecifically from the pro-S position. Arg-116, with its positive charge, supports the transfer of an electron from reduced FMN to dioxygen.
Group members
Mgr. Vojtěch Sedláček Ph.D. – lecturer II
Ph.D. candidates: Ivo Fukala, Martin Kryl
Ph.D. alumni: Pavel Bouchal, Martin Hubálek, Marek Koutný, Michal Kuňák, Jiří Mazoch, Nikola Ptáčková, Vladimír Rotrekl, Vojtěch Sedláček, Jiří Zouhar
International cooperation
- IKBM, Norwegian University of Life Sciences (NMBU) – prof. Åsa Helena Frostegård
- Systems Biology Lab, Vrije Universiteit Amsterdam, Netherlands – dr. Rob van Spanning