Molekularbiologische und biochemische Untersuchungen der Biosynthese und Metabolisierung von cyclischen Dipeptiden in Actinobacteria

2,5-Diketopiperazine sind die Grundgerüste von einigen bereits zugelassenen Arzneistoffen mit unterschiedlichen Indikationen und stellen ebenso wichtige Grundstrukturen für weitere potentielle Wirkstoffe dar. Das Grundgerüst der 2,5-Diketopiperazine kann unter anderem von Cyclodipeptid-synthasen (CD...

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Bibliographic Details
Main Author: Brockmeyer, Kirsten
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
Online Access:PDF Full Text
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2,5-Diketopiperazines are the basic structures of some already approved drugs with different indications and therefore represent important skeletons for new potential biological active structures. This basic structure can be formed by cycliodipeptide synthases (CDPSs). It has been postulated that special motifs in the active center allow a prediction of possibly formed cyclic dipeptides. In this work, a cyclodipeptide synthase from Nocardiopsis prasina named CDPS-Np was analyzed. According to the motives in the active center, this CDPS is proposed to produce phenylalanine-containing cyclic dipeptides. After experimental proof, CDPS-Np was shown to produce tyrosine-containing cyclic dipeptides with cyclo(L-Tyr-L-Phe) as the main and cyclo(L-Tyr-L-Tyr), cyclo(L-Tyr-L-Met) and cyclo(L-Tyr-L-Leu) or cyclo(L-Tyr-L-Ile) as minor products. These results provided evidence that this kind of prediction allows to become a hint of possible product formation, but an experimental proof is necessary. Due to the deviation of the actual from the expected product formation, motifs in the active center were analyzed. The substrate specificity should be changed by site-directed mutagenesis in the two binding pockets P1 and P2. For this purpose, various mutations were first introduced in position Thr82 and Tyr196 in binding pocket P1 of CDPS-Np. The original intention to change the substrate specificity could not be achieved by these experiments. However, an 8-fold increase in product formation of cyclo(L-Tyr-L-Phe) and 10-fold of cyclo(L-Tyr-L-Tyr) were detected in the case of the mutant CDPS-Np_T82V_Y196 compared to that of the wild type. Site-directed mutagenesis in the binding pocket P2 also did not change the product spectrum. While the CDPSs are able to form the basic structure of potential active agents, different enzymes are able to modify these structures. Methyltransferases are able to attach a methyl-residue to either C- or O-atoms. Cytochrome P450 enzymes are for example able to bild C-C-bonds and to introduce hydroxy groups or double bonds. In this work, cyclodipeptide oxidases (CDOs) were analyzed, which introduce double bonds at positions 3 and 6 of 2,5-diketopiperazine skeleton. CDOs consist of two subunits, which are only together active for introduction of double bonds. Until now only two CDOs has been analyzed in details. AlbA and AlbB from Streptomyces noursei and Ndas_1146 and Ndas_1147 from Nocardiopsis dassonvillei. The third CDO named CDOA-Np and CDOB-Np from Nocardiopsis prasina has been analyzed in this work. For this purpose, the overlapping nucleotide sequence was cloned into a vector for overproduction of CDOA-Np and CDOB-Np in E. coli. By identifying the enzymatic products of a biotransformation of the natural substrate cyclo(L-Tyr-L-Phe) in E. coli, the functionality of CDOA-Np and CDOB-Np could be confirmed. Furthermore, the activity of His6-CDOA-Np and CDOB-Np-His6 was also shown. For futher investigations, the separated genes of the cyclodipeptide oxidases from Nocardiopsis prasina and Nocardiopsis dassonvillei were already cloned into different expression vectors for overproduction in E. coli. This requires the optimization of overproduction and purification of CDOA-Np-His6, CDOB-Np-His6, Ndas-1146-His6 and Ndas-1147-His6. Furthermore, the cyclodipeptide oxidase from Nocardiopsis prasina was compared with the alrady known cyclodipeptide oxidases from Nocardiopsis dassonvillei and Streptomyces noursei. For this purpose, the overlapping nucleotide sequence from Streptomyces noursei was cloned into an expression vector for overproduction of AlbA and AlbB in E. coli. Subsequently, the biotransformation of various cyclic dipeptides by CDOA-Np/ CDOB-Np, Ndas_1146/Ndas_1147 as well as AlbA/AlbB was investigated. After identification of the enzymatic products by LC-MS, these results were compared. In this context it was the first time that these three cyclodipeptide oxidases has been compared with each other. These enzymes share similar, but not identical behaviors. The mechanism of the CDO reaction is unknown. A direct dehydration, α-hydroxylation followed by loss of water or imine formation and a following isomerization are possible scenarios. The mechanism was analyzed by biotransformation of deuterated cyclo(L-Tyr-L-Phe) using CDOA-Np/CDOB-Np from Nocardiopsis prasina, Ndas_1146/Ndas_1147 from Nocardiopsis dassonvillei and AlbA/AlbB from Streptomyces noursei in E. coli. After analysis of the products by LC-MS, no product indicating an imine-intermediate were detected. These results suggest a direct dehydration as plausible mechanism of the reaction.