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The basis of the structural-functional study of selected enzymes from the 4-alkyl- L-proline pathway
Zachovalová, Veronika ; Zdvořáková, Lucie (advisor) ; Košek, Dalibor (referee)
4-alkyl-L-proline derivate (APD) is building block of some important, bioactive specialized metabolites produced by Actinobacteria. It is found, for example, in the structure of lincosamide antibiotic lincomycin, bacterial hormone hormaomycin having antimicrobial activity, the antituberculosis compound griselimycin and pyrrolo-1,4-benzodiazepins (PBD) which have anticancer effects. APDs, or their precursors, are formed from L-leucine or L-tyrosin in a unique biosynthetic pathway consisting of up to six homologous proteins named Apd1 - Apd6. The first part of this thesis lays the foundation for a crystallization study of SAM-dependent methyltransferase Apd3 from the biosynthesis of lincomycin - the protein LmbW, in order to elucidate the identity of its natural substrate, which remains up to date a subject of scientific discussions. A purification method providing LmbW protein of sufficient quantity, quality and purity for crystallization was optimized. Subsequently the crystallization conditions allowing crystal growth were defined. The second part of the thesis deals with the elucidation of molecular basis of different reaction specificity of homologous F420H2-dependent oxidoreductases, Apd6. They catalyse reduction of identical substrate with two conjugated double bonds, however, some of them...
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