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

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Duis id rutrum mi, eget efficitur mi. Nullam facilisis porta erat in accumsan. Suspendisse convallis finibus arcu, et euismod quam laoreet eget. Donec mollis dui tellus, ut hendrerit massa iaculis quis. Etiam accumsan lectus commodo, rhoncus dolor nec, pretium sapien.

MUNI | orcID | ResearchGate

About Igor Kučera

Contact information:

  • Office – C05/234
  • Phone – 549 49 5392
  • E-mail – ikucera@chemi.muni.cz

Teaching

  • C8112 Enzyme biotechnology
  • C8140 Bioenergetics
  • C8150 Bioenergetics - seminar
  • C5340 Nonequilibrium systems
  • C8160 Enzymology
  • C8170 Enzymology - seminar

Education

  • 1998 – professor of Biochemistry, SCI MU
  • 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). 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

Sedláček V, Kučera I. Arginine-95 is important for recruiting superoxide to the active site of the FerB flavoenzyme of Paracoccus denitrificans. FEBS Lett. 2019; 593(7): 697-702. doi: 10.1002/1873-3468.13359.

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.

***

Sedláček V, Kučera I. Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans. Mol. Microbiol. 2019; 112(1):166-183. doi: 10.1111/mmi.14260.

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.

***

Sedláček V, Kučera I. Modifications of the Aerobic Respiratory Chain of Paracoccus Denitrificans in Response to Superoxide Oxidative Stress. Microorganisms. 2019; 7(12):640. doi: 10.3390/microorganisms7120640.

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.

***

Kučera I, Sedláček V. Involvement of the cbb3-Type Terminal Oxidase in Growth Competition of Bacteria, Biofilm Formation, and in Switching between Denitrification and Aerobic Respiration. Microorganisms. 2020; 8(8):1230. doi: 10.3390/microorganisms8081230.

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.

***

Sedláček V, Kryl M, Kučera I. The ArsH Protein Product of the Paracoccus denitrificans ars Operon Has an Activity of Organoarsenic Reductase and Is Regulated by a Redox-Responsive Repressor. Antioxidants. 2022; 11(5):902. doi: 10.3390/antiox11050902.

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.

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

You are running an old browser version. We recommend updating your browser to its latest version.

More info