Obiettivo
Mammalian quinoproteins are involved in the biosynthesis and degradation of compounds (eg, noradrenaline) regulating important physiological processes. However, although they are very important targets from a pharmacological point of view, relatively little is known about these enzymes with respect to structure and enzymology.
X-ray diffraction of crystals of methylamine dehydrogenase, sequencing of the gene and protein, and characterisation of the cofactor in the enzyme, have led to the elucidation of the 3-dimensional structure of the first quinoprotein.
Enzymes with a quinone cofactor (quinoproteins) play a role in incomplete microbial oxidations (vinegar and gluconid acid production), in regulation of cell proliferation and collagen formation in mammals. Furthermore, they have potentials for application in production of fine chemicals and development of amperometric biosensors. For these reasons, insight into the catalytic mechanism of enzymes with these novel quinone cofactors could provide tools for application and regulation of quinoproteins. Methylamine dehydrogenase from the bacterium Thiobacillus versutus was used as a model enzyme for that purpose.
Methylamine dehydrogenase from Thiobacillus versutus was obtained as a homogeneous preparation. From the molecular weight and studies on the subunits it appears that it has an alpha2beta2 structure. Homogeneous preparations of the alpha subunit-and beta subunit were obtained by denaturing the enzyme with guanidiniumhydrochloride, followed by gel filtration chromatography. By using specific proteases, peptides were obtained by high performance liquid chromatography (HPLC). A number of peptides were sequenced, providing information to construct probes to clone the genes, to elucidate the amino acid sequence around the attachment sites of the cofactor to the protein chain, and to support the work on the 3-dimensional structure elucidation. A piece of deoxyribonucleic acid (DNA) was isolated containing the gene for the beta subunit. Subsequently it was found that it also contains the gene for the alpha subunit with an intermediate piece of unknown function and sequence. From the sequence of the gene for the beta subunit, 2 tryptophans, representing the building blocks of the cofactor, were found at the same positions as have been found for the enzyme from Methylobacterium extorquens. Together with results from Raman spectroscopy of the enzyme and nuclear magnetic resonance (NMR) spectroscopy of the isolated, derivatised cofactor, this proves that the cofactor in t he enzyme from Th versutus is also tryptophan trytophyl quinone TTQ. Although just as PQQ, TTQ has an o-quinone group, the structure of this cofactor is unprecedented, demonstrating that novel cofactors can still be found.
Methylamine dehydrogenase from Thiobacillus versutus was obtained as a homogeneous preparation. From the molecular weight and studies on the subunits, it appears that it has an alpha 2 beta 2 structure. Homogeneous preparations of the alpha and beta subunits were obtained and by using specific proteases, peptides prepared by high pressure liquid chromatography. A number of peptides were sequenced, providing information to construct probes to clone the genes, to elucidate the amino acid sequence around the attachment sites of the cofactor to the protein chain, and to support the work on the 3-dimensional structure elucidation. Results prove that the cofactor in the enzyme from Th versutus is also tryptophan tryptophyl quinone (TTQ). Attempts to crystallyze the enzyme were successful which led to the first 3-dimensional structure elucidation of a quinoprotein enzyme. The use of crystals treated with inhibitors has shown that the reactive carbonyl group is situated at the C6 carbon atom.
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CORDIS classifica i progetti con EuroSciVoc, una tassonomia multilingue dei campi scientifici, attraverso un processo semi-automatico basato su tecniche NLP. Cfr.: Il Vocabolario Scientifico Europeo.
CORDIS classifica i progetti con EuroSciVoc, una tassonomia multilingue dei campi scientifici, attraverso un processo semi-automatico basato su tecniche NLP. Cfr.: Il Vocabolario Scientifico Europeo.
- scienze naturali scienze biologiche genetica DNA
- scienze naturali scienze biologiche zoologia mammologia
- scienze naturali scienze chimiche chimica organica ammine
- scienze naturali scienze biologiche biochimica biomolecole proteine enzimi
- scienze naturali scienze fisiche ottica spettroscopia
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