03 Fakultät Chemie
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Item Open Access Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli(2021) Hunold, Andreas; Escobedo-Hinojosa, Wendy; Potoudis, Elsa; Resende, Daniela; Farr, Theresa; Syrén, Per-Olof; Hauer, BernhardPhenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile.Item Open Access Active-site loop variations adjust activity and selectivity of the cumene dioxygenase(2021) Heinemann, Peter M.; Armbruster, Daniel; Hauer, BernhardActive-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.Item Open Access Loops und Tunnel : unterschätzte Elemente in Enzymen(2020) Heinemann, Peter M.; Rapp, Lea R.; Hauer, BernhardIn enzymes, the active site is the location where substrates are chemically converted. If this site is deeply buried within the protein, substrates must pass not only through the body of the protein via a tunnel, but also flexible, site decorating loops to access the active site. These elements can act as filters that influence on both substrate specificity and activity. Identifying and understanding how they exert such control has been of growing interest over the past several years.Item Open Access Enhanced semi‐preparative biotransformation of cumene dioxygenase : from analytical scale to product isolation(2023) Schelle, Jona T.; Lepoittevin, William; Hauer, BernhardScale‐up of oxygenase catalyzed reactions is often challenging due to the limited oxygen mass transfer in aqueous solutions. To overcome such limitation, we studied different scale‐up conditions using recombinant resting cells of E. coli JM109(DE3), harboring the cumene dioxygenase of Pseudomonas fluorescens IP01, for the dihydroxylation of naphthalene to (1R,2S)‐cis‐1,2‐dihydro‐1,2‐naphthalenediol. Thereby, vigorous stirring of the biotransformation in a 2 L round bottom flask in combination with an oxygen‐enriched headspace exhibited outstanding product formation after 1 h. Furthermore, the enhanced setup was used for the cumene dioxygenase catalyzed biosynthesis of 240 mg of valuable (+)‐trans‐carveol from (R)‐(+)‐limonene, demonstrating the application of our workflow for volatile compounds.Item Open Access Modifizierte Enzyme ermöglichen die selektive N‐Alkylierung von Pyrazolen unter Verwendung einfacher Halogenalkane(2021) Bengel, Ludwig L.; Aberle, Benjamin; Egler‐Kemmerer, Alexander‐N.; Kienzle, Samuel; Hauer, Bernhard; Hammer, Stephan C.Die selektive Alkylierung von Pyrazolen ist eine Herausforderung in der Chemie und könnte die Synthese wichtiger Moleküle vereinfachen. In dieser Arbeit berichten wir über eine katalysatorgesteuerte Alkylierung von Pyrazolen durch eine cyclische Kaskadenreaktion mit zwei Enzymen. In diesem enzymatischen System nutzt ein promiskuitives Enzym Halogenalkane als Ausgangsstoffe, um nicht-natürliche Analoga des Cosubstrats S-Adenosyl-l-Methionin zu synthetisieren. Ein zweites engineertes Enzym überträgt die Alkylgruppen in einer hochselektiven C-N-Bindungsknüpfung auf das Pyrazol-Substrat. Das Cosubstrat wird regeneriert und nur in katalytischen Mengen eingesetzt. Für das Enzym-Engineering wurde eine computerbasierte Methode verwendet, um eine Mutantenbibliothek in silico zu entwickeln. In einer Runde von Mutagenese und Screening wurde somit eine promiskuitive Methyltransferase in eine kleine Pyrazol-alkylierende Enzymfamilie umgewandelt. Mit diesem bienzymatischen System konnte die Alkylierung von Pyrazolen (Methylierung, Ethylierung, Propylierung) mit bislang unerreichter Regioselektivität (>99 %), Regiodivergenz und in einem ersten Beispiel in präparativem Maßstab gezeigt werden.