Graz University of Technology Austria Institute of Biochemistry ...
Graz University of Technology Austria Institute of Biochemistry ... Graz University of Technology Austria Institute of Biochemistry ...
Luciferase and LuxF The emission of light by biological species (bioluminescence) is a fascinating process found in diverse organisms such as bacteria, funghi, insects, fish, limpets and nematodes. In all cases the bioluminescent process is based on a chemiluminescent reaction in which the chemical energy is (partially) transformed into light energy ("cold light"). All bioluminescent processes require a luciferase, i.e. an enzyme catalyzing the chemiluminescent reaction, and a luciferin, which can be considered a coenzyme. During the bioluminescent reaction the luciferin is generated in an excited state and serves as the emitter of light energy. In our laboratory, we are interested in the bioluminescence of marine photobacteria. In these bacteria, luciferase is composed of an alpha/beta-heterodimeric protein, which binds reduced flavinmononucleotide (FMN) as the luciferin. The reduced FMN reacts with molecular dioxygen to a hydroperoxide intermediate with subsequent oxidation of a long-chain fatty aldehyde (e.g. tetradecanal) to the corresponding fatty acid (e.g. myristic acid). During this oxidation process, an excited flavin intermediate is generated which emits light. Some marine photobacteria possess an additional protein called LuxF which was found in complex with a myristylated flavin derivative where the C-3 atom of myristic acid is covalently attached to the 6-position of the flavin ring system. It was postulated that this flavin adduct is generated in the luciferase catalyzed bioluminescent reaction. Furthermore, it was speculated that LuxF sequesters the myristylated flavin adduct in order to prevent inhibition of the bioluminescent reaction. However, both hypotheses have not been tested on a biochemical or physiological level yet. Hence, in this study we will design and perform experiments to examine the putative generation of myristylated FMN through the luciferase reaction (thesis project of Thomas Bergner supported by Steve Stipsits) Structure of a LuxF dimer in the absence (red) and presence (blue) of the myristylated flavin derivative (pdb code 1NFP) Nikkomycin biosynthesis Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, insecticidal and acaricidal properties due to their strong inhibition of chitin synthase. Nikkomycins are promising compounds in the cure of the immunosuppressed, such as AIDS patients, organ transplant recipients and cancer patients undergoing chemotherapy. Nikkomycin Z (R 1 = uracil & R 2 = OH, see below) is currently in clinical trial for its antifungal activity. Structurally, nikkomycins can be classified as peptidyl nucleosides containing two 10
unusual amino acids, i.e. hydroxypyridylhomothreonine (HPHT) and aminohexuronic acid with an N-glycosidically linked base: Although the chemical structures of nikkomycins have been known since the 1970s, only a few biosynthetic steps have been investigated in detail. The steps leading to the synthesis of aminohexuronic acid are unclear. Originally, it was hypothesized that the aminohexuronic acid moiety is generated by addition of an enolpyruvyl moiety from phosphoenolpyruvate (PEP) to either the uridine or the 4-formyl-4-imidazolin-2-one analog at the 5’-position of ribose. This step is then followed by rather speculative modifications to yield the aminohexuronic acid precursor. In contrast to this hypothesis, we could recently demonstrate that UMP rather than uridine serves as the acceptor for the enolpyruvyl moiety, a reaction catalyzed by an enzyme encoded by a gene of the nikkomycin operon termed nikO. Furthermore, we could demonstrate that it is attached to the 3’- rather than the 5’-position of UMP. These results are very intriguing since none of the nikkomycins synthesized possess an enolpyruvyl group in this position of the sugar moiety. Hence, it must be concluded that the resulting 3’-enolpyruvyl-UMP is subject to rearrangement reactions where the enolpyruvyl is detached from its 3’-position and transferred to the 5’-position of the ensuing aminohexuronic acid moiety. Co-crystallization of NikO with fosfomycin yielded rod – like crystals diffracting up to 2.5 Å. A synchrotron dataset was measured at the Swiss Light Source and the structure was solved by molecular replacement using UDP-N-acetylglucosamine enolpyruvyl transferase (PDB code: 2rl1) as a model. Two molecules were found in the asymmetric unit exhibiting an inverse α,β-barrel fold with helices forming the tightly packed core and sheets shielding the hydrophobic core from the solvent: Eech chain is comprised of two inverse α,β-barrel subunits, which are connected by a hinge region. The final structure was refined to final R/R free values of 17% and 19%, respectively. Crystallization trials of NikO in the presence of its product, 3’-EP-UMP, are currently under way. 11
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Luciferase and LuxF<br />
The emission <strong>of</strong> light by biological species (bioluminescence) is a fascinating process found<br />
in diverse organisms such as bacteria, funghi, insects, fish, limpets and nematodes. In all cases<br />
the bioluminescent process is based on a chemiluminescent reaction in which the chemical<br />
energy is (partially) transformed into light energy ("cold light"). All bioluminescent processes<br />
require a luciferase, i.e. an enzyme catalyzing the chemiluminescent reaction, and a luciferin,<br />
which can be considered a coenzyme. During the bioluminescent reaction the luciferin is<br />
generated in an excited state and serves as the emitter <strong>of</strong> light energy. In our laboratory, we<br />
are interested in the bioluminescence <strong>of</strong> marine photobacteria. In these bacteria, luciferase is<br />
composed <strong>of</strong> an alpha/beta-heterodimeric protein, which binds reduced flavinmononucleotide<br />
(FMN) as the luciferin. The reduced FMN reacts with molecular dioxygen to a hydroperoxide<br />
intermediate with subsequent oxidation <strong>of</strong> a long-chain fatty aldehyde (e.g. tetradecanal) to<br />
the corresponding fatty acid (e.g. myristic acid). During this oxidation process, an excited<br />
flavin intermediate is generated which emits light. Some marine photobacteria possess an<br />
additional protein called LuxF which was found in complex with a myristylated flavin<br />
derivative where the C-3 atom <strong>of</strong> myristic acid is covalently attached to the 6-position <strong>of</strong> the<br />
flavin ring system. It was postulated that this flavin adduct is generated in the luciferase<br />
catalyzed bioluminescent reaction. Furthermore, it was speculated that LuxF sequesters the<br />
myristylated flavin adduct in order to prevent inhibition <strong>of</strong> the bioluminescent reaction.<br />
However, both hypotheses have not been tested on a biochemical or physiological level yet.<br />
Hence, in this study we will design and perform experiments to examine the putative<br />
generation <strong>of</strong> myristylated FMN through the luciferase reaction (thesis project <strong>of</strong> Thomas<br />
Bergner supported by Steve Stipsits)<br />
Structure <strong>of</strong> a LuxF dimer in the absence (red) and presence (blue) <strong>of</strong> the myristylated flavin<br />
derivative (pdb code 1NFP)<br />
Nikkomycin biosynthesis<br />
Nikkomycins are produced by several species <strong>of</strong> Streptomyces and exhibit fungicidal,<br />
insecticidal and acaricidal properties due to their strong inhibition <strong>of</strong> chitin synthase.<br />
Nikkomycins are promising compounds in the cure <strong>of</strong> the immunosuppressed, such as AIDS<br />
patients, organ transplant recipients and cancer patients undergoing chemotherapy.<br />
Nikkomycin Z (R 1 = uracil & R 2 = OH, see below) is currently in clinical trial for its antifungal<br />
activity. Structurally, nikkomycins can be classified as peptidyl nucleosides containing two<br />
10
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