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Titel:Molekularbiologische und biochemische Untersuchungen zu Prenyltransferasen und NRPS-ähnlichen Enzymen aus Ascomyceten
Autor:Tarcz, Sylwia Magdalena
Weitere Beteiligte: Li, Shu-Ming (Prof. Dr.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0563
DOI: https://doi.org/10.17192/z2014.0563
URN: urn:nbn:de:hebis:04-z2014-05632
DDC: Medizin, Gesundheit
Titel(trans.):Molecular biological and biochemical investigations on prenyltransferases and NRPS-like enzymes from ascomycetes
Publikationsdatum:2015-07-28
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Site Directed Mutagenesis, O, Secondary Metabolism, Aspergillus terreus, Indolalkaloide, Sekundärstoffwechsel, Aspergillus fumigatus, Prenyltransferases, Indole Alkaloids, Peptidsynthetasen, Prenyltransferasen, Chaetomium globosum

Zusammenfassung:
Um ihr eigenes Überleben zu sichern und sich Vorteile gegenüber anderen Bewohnern ihrer Umwelt zu verschaffen, haben Mikroorganismen die Fähigkeit entwickelt, bioaktive Sekundärmetabolite zu produzieren. Diese kann sich der Mensch als Antibiotika, Immunsuppressiva oder als Leitsubstanzen für die Entwicklung neuer Wirkstoffe wie HIV-Proteaseinhibitoren oder Insulinmimetika ebenfalls zu Nutze machen. Damit stellen sie eine wichtige Quelle potentiell nutzbarer Wirkstoffe dar. Große Fortschritte wurden in den vergangenen Jahrzehnten auf dem Gebiet der Erforschung der für die Biosynthese dieser Metabolite notwendigen mikrobiellen Werkzeuge gemacht. Einerseits sind nichtribosomale Peptidsynthetasen und Polyketidsynthasen weit verbreitete Enzyme, die in viele Biosynthesen von Grundstrukturen involviert sind und aufgrund ihrer modularen Struktur viel Spielraum für Manipulationen durch die Synthetische Biologie bieten. Andererseits können Sekundärmetabolite durch Modifikationsenzyme (tailoring enzymes) wie z. B. Prenyltransferasen weiter verarbeitet werden, was ihre biologische Aktivität häufig erhöht. Um diese Enzyme gezielt einsetzen zu können, ist es von essentieller Bedeutung, ihre Struktur und Funktion zu untersuchen und zu verstehen. Zahlreiche prenylierte Bisindolylbenzochinone aus der Gruppe der prenylierten Indol-alkaloide konnten bereits aus Ascomyceten isoliert werden, biochemische Informationen waren bisher allerdings nur für die Biosynthese von Terrechinon A in Aspergillus nidulans verfügbar. Im Rahmen der vorliegenden Dissertation konnten die putativen Prenyltransferasegene astPT und ATEG_00702 (EAU39348) aus Aspergillus terreus heterolog in Escherichia coli exprimiert und anschließend bis zur Homogenität aufgereinigt werden. Das rekombinante Protein AstPT katalysierte in Anwesenheit von Dimethylallyldiphosphat (DMAPP) die N- und C-Prenylierung von Asterrichinon D (AQ D). Als enzymatische Produkte konnten zwei mono- und zwei diprenylierte Substanzen isoliert werden, deren Strukturen mittels NMR-Spektroskopie und Massenspektrometrie aufgeklärt wurden. Wie andere Prenyltransferasen der DMATS-Superfamilie war AstPT in seiner Katalyse unabhängig von Metallionen und folgte der Michaelis-Menten-Kinetik mit einer Michaelis-Konstante von 463 µM und einer Wechselzahl von 0,16 1/s für AQ D bzw. von 33,5 µM und 0,02 1/s für DMAPP. Besonderheiten von AstPT sind die Einführung von Prenyleinheiten an zwei verschiedenen Positionen des Substrates und zum anderen die hohe Spezifität für seine aromatischen Substrate. AstPT akzeptierte weder Aminosäuren noch cyclische Dipeptide, wie sonst typisch für Prenyltransferasen der DMATS-Superfamilie. Hingegen akzeptierte sie vier Hydroxyxanthonderivate als Substrate und katalysierte O-Prenylierungen in Anwesenheit von DMAPP, Geranyl- (GPP) und Farnesyldiphosphat (FPP), was bisher nur für ein Enzym der DMATS-Superfamilie beobachtet werden konnte. Die Michaelis-Konstante für das beste Xanthonderivat wurde mit 17,3 µM bestimmt, allerdings war die Wechselzahl für AQ D um das 160-fache höher als für das Xanthonderivat. EAU39348 konnte AQ D nicht umsetzen, mit Didemethylasterrichinon D (DDAQ D) als aromatisches Substrat war hingegen die Bildung von mehreren Produkten zu beobachten. Die langsame Umsetzung von AQ D durch AstPT und die alleinige Umsetzung von DDAQ D durch EAU39348 deuten auf eine bevorzugte Produktion von DDAQ D-Derivaten in Aspergillus terreus hin, die in deren höherer Bioaktivität begründet sein könnte. Ein drittes putatives Prenyltransferasegen, CHGG_03684, konnte aus Chaetomium globosum kloniert und in verschiedenen Expressionssystemen getestet werden. Unter diesen Bedingungen konnte jedoch kein lösliches Protein erhalten werden. Nach Solubilisierung der Protein-Einschlusskörper mittels Harnstoff wurde eine Renaturierung durch Dialyse versucht. Mit dem erhaltenen Protein und AQ D konnte in Anwesenheit von DMAPP keine Umsetzung beobachtet werden. Des Weiteren wurden vier putative, NRPS-ähnliche Gene aus Aspergillus terreus bzw. Chaetomium globosum mittels PCR amplifiziert. ATEG_02004, ATEG_03563 und CHGG_03687 konnten in den Expressionsvektor pJW24 kloniert werden, der pyrG als Selektionsmarker für die anschließende Transformation trägt. Das Konstrukt mit ATEG_02004 konnte erfolgreich in Aspergillus niger AB 1.13 eingebracht und die Integration in Genom mittels PCR nachgewiesen werden. Die Untersuchung der Sekundärmetabolit-produktion der Transformanten führte allerdings nicht zur Identifizierung der Funktion von ATEG_02004. In einem weiteren Projekt innerhalb dieser Arbeit wurde die Bedeutung verschiedener Aminosäuren in FtmPT1 untersucht, um den auf der Enzymstruktur basierten Reaktions-mechanismus zu bestätigen. Durch zielgerichtete Mutagenese mittels PCR und anschließende Überproduktion und Aktivitätstests konnte die Beteiligung des Glutamatrests 102 an der Stabilisierung des Indolstickstoffs über Wasserstoffbrückenbindungen und seine Rolle als Protonenakzeptor während der Katalyse gezeigt werden. Der Austausch von Glycin 115 führte zur Änderung der Prenylierungsposition von C 2 nach C 3 unter Entstehung eines Hexahydropyrroloindols. Dies konnte sowohl für das natürliche Substrat von FtmPT1, Brevianamid F, als auch für verschiedene cyclische Dipeptide nachgewiesen werden. Damit wurde die essentielle Rolle von G115 in der Prenylierung in FtmPT1 aufgezeigt. Mutationen der entsprechenden Aminosäure in CdpC3PT, A115, senkten nur die Aktivität. Mit dem Austausch von Tyrosin 205 gegen Phenylalanin konnte die Akzeptanz von sechs Hydroxynaphthalinderivaten gegenüber dem Wildtypprotein verbessert werden.

Summary:
Microorganisms developed the capability to produce bioactive secondary metabolites to ensure their own viability and get evolutionary advantages. These metabolites can be used as antibiotics or immunosuppressive drugs, but also as lead structures for the development of new drugs like HIV protease inhibitors or insulin mimetics. Therefore, microorganisms are an important source of potentially active pharmaceutical ingredients. Significant progress has been achieved in the past decades in the research on the biosynthesis of these compounds. On the one hand, nonribosomal peptide synthetases and polyketide synthases are common enzymes involved in the biosynthesis of the backbones of numerous metabolites and their module-based structure represents a target for manipulations with the means of synthetic biology. On the other hand, secondary metabolites can be further modified by tailoring enzymes like prenyltransferases which often leads to a higher biological activity. It is of particular importance to investigate these structures and to understand their functions in order to make use of those enzymes. Many prenylated bisindolylbenzoquinones belonging to prenylated indole alkaloids have been isolated from ascomycetes, but biochemical information has been available only for the biosynthesis of terrequinone A in Aspergillus nidulans prior to this thesis. In this thesis the putative prenyltransferase genes astPT and ATEG_00702 (EAU39348) from Aspergillus terreus were expressed heterologously in Escherichia coli and subsequently purified to near homogeneity. The recombinant protein AstPT catalysed the N- and C-prenylation of asterriquinone D in the presence of dimethylallyldiphosphate (DMAPP). The enzyme products, two mono- and two diprenylated compounds, were isolated and their structure was elucidated by NMR spectroscopy and mass spectrometry. As other prenyltransferases from the DMATS-superfamily, AstPT was not dependent on the presence of metal ions for its catalytic activity. The reaction apparently followed Michaelis-Menten kinetics and the Michaelis constant was determined at 463 µM with a turnover number at 0.16 s-1 for AQ D and at 33.5 µM and 0.02 s 1 for DMAPP, respectively. Special features of AstPT were the introduction of prenyl moieties at two distinct sites of the substrate and its high specificity towards its aromatic substrate. It accepted neither amino acids nor cyclic dipeptides, which are typical substrates of prenyltransferases of the DMATS-superfamily. However, it accepted four hydroxyxanthone derivatives as substrates and catalysed O-prenylations in the presence of DMAPP but also from geranyl- (GPP) and farnesyldiphosphate (FPP). This phenomenon has only been observed for one enzyme from this group before. The best accepted xanthone derivative had a Michaelis constant at 17.3 µM, but the turnover number for AQ D was 160 times higher than for this hydroxyxanthone. EAU39348 was not able to convert AQ D. Using didemethylasterriquinone D (DDAQ D) as aromatic substrate, the formation of several products was observed. The slow conversion of AQ D by AstPT and the exclusive acceptance of DDAQ D by EAU39348 may indicate that the production of DDAQ D derivatives is preferred in Aspergillus terreus. The reason for this might be the stronger biological activities of DDAQ D derivatives in comparison to AQ D derivatives. A third putative prenyltransferase gene, CHGG_03684, was cloned from Chaetomium globosum and overproduction was tested in different expression systems. Unfortunately, no soluble protein was obtained under these conditions. An attempt of renaturation via dialysis was made after solubilisation of inclusion bodies with urea, but no activity was observable after incubation with AQ D and DMAPP. Furthermore, four putative NRPS-like genes from Aspergillus terreus and Chaetomium globosum were amplified via PCR. ATEG_02004, ATEG_03563 and CHGG_03687 were cloned into the expression vector pJW24 containing pyrG as a selection marker for subsequent transformations. The construct with ATEG_02004 was successfully trans-ferred into Aspergillus niger AB 1.13 and the genomic integration was confirmed via PCR. The function of ATEG_02004 could not be identified by investigation of the produced secondary metabolites. In addition, the importance of several amino acid residues in FtmPT1 was studied in this thesis to support the proposed reaction mechanism, which is based on the enzyme structure. The involvement of glutamate 102 in the stabilization of the indole nitrogen and its role as proton acceptor during catalysis was shown in activity assays after site-directed mutagenesis and overproduction of the proteins. Exchange of glycine 115 for threonine led to a shift of the prenylation position from C 2 to C 3 and the introduction of a hexahydropyrroloindole. This was observed by using the natural substrate brevianamide F as well as different cyclic dipeptides. These results revealed the essential role of G115 in the prenylation of FtmPT1. Mutation of the corresponding amino acid in CdpC3PT did not have any effect on the prenylation position and simply decreased the activity. The exchange of tyrosine 205 for phenylalanine increased the acceptance of six hydroxynaphthaline derivatives.

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