Carolin Bisterfeld

Trading off stability against activity in extremophilic aldolases.

Understanding enzyme stability and activity in extremophilic organisms is of great biotechnological interest, but many questions are still unsolved. Using 2-deoxy-D-ribose-5-phosphate aldolase (DERA) as model enzyme, we have evaluated structural and functional characteristics of different orthologs from psychrophilic, mesophilic and hyperthermophilic organisms. We present the first crystal structures of psychrophilic DERAs, revealing a dimeric organization resembling their mesophilic but not their thermophilic counterparts. Conversion into monomeric proteins showed that the native dimer interface contributes to stability only in the hyperthermophilic enzymes. Nevertheless, introduction of a disulfide bridge in the interface of a psychrophilic DERA did confer increased thermostability, suggesting a strategy for rational design of more durable enzyme variants. Constraint network analysis revealed particularly sparse interactions between the substrate pocket and its surrounding α-helices in psychrophilic DERAs, which indicates that a more flexible active center underlies their high turnover numbers.

Chemoenzymatic Total Synthesis of the Proposed Structures of Putaminoxins B and D

Different enzymatic and nonenzymatic approaches were tested and compared to afford enantiopure homoallylic and allylic alcohols as building blocks in a total synthesis showcase. Thereby, highly enantioselective alcohol dehydrogenases and the P450 BM3 monooxygenase variant A74G L188Q were compared to classical asymmetric reagent-controlled allyl additions. Thus, the first total syntheses of the proposed structures for putaminoxins B/D and their respective enantiomers were accomplished. Detailed spectroscopic analysis of the newly synthesized compounds unraveled a discrepancy with respect to the reported structures of putaminoxins B/D. Furthermore, it was demonstrated that total synthesis is generally required for unequivocal assignment of configuration, because purely comparative NMR studies and judgment by analogy can lead to false predictions.

Immobilization of 2-Deoxy-d-ribose-5-phosphate Aldolase in Polymeric Thin Films via the Langmuir-Schaefer Technique

A synthetic protocol for the fabrication of ultrathin polymeric films containing the enzyme 2-deoxy-d-ribose-5-phosphate aldolase from Escherichia coli (DERAEC) is presented. Ultrathin enzymatically active films are useful for applications in which only small quantities of active material are needed and at the same time quick response and contact times without diffusion limitation are wanted. We show how DERA as an exemplary enzyme can be immobilized in a thin polymer layer at the air-water interface and transferred to a suitable support by the Langmuir-Schaefer technique under full conservation of enzymatic activity. The polymer in use is a poly(N-isopropylacrylamide-co-N-2-thiolactone acrylamide) (P(NIPAAm-co-TlaAm)) statistical copolymer in which the thiolactone units serve a multitude of purposes including hydrophobization of the polymer, covalent binding of the enzyme and the support and finally cross-linking of the polymer matrix. The application of this type of polymer keeps the whole approach simple as additional cocomponents such as cross-linkers are avoided.

Redesigning Aldolase Stereoselectivity by Homologous Grafting

The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity.

Biocatalytically Active Thin Films via Self-Assembly of 2-Deoxy-d-ribose-5-phosphate Aldolase–Poly(N-isopropylacrylamide) Conjugates

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a biocatalyst that is capable of converting acetaldehyde and a second aldehyde as acceptor into enantiomerically pure mono- and diyhydroxyaldehydes, which are important structural motifs in a number of pharmaceutically active compounds. However, substrate as well as product inhibition requires a more-sophisticated process design for the synthesis of these motifs. One way to do so is to the couple aldehyde conversion with transport processes, which, in turn, would require an immobilization of the enzyme within a thin film that can be deposited on a membrane support. Consequently, we developed a fabrication process for such films that is based on the formation of DERA-poly(N-isopropylacrylamide) conjugates that are subsequently allowed to self-assemble at an air-water interface to yield the respective film. In this contribution, we discuss the conjugation conditions, investigate the interfacial properties of the conjugates, and, finally, demonstrate a successful film formation under the preservation of enzymatic activity.

Cyclopropyl Lactone-Containing Marine Oxylipins

Abstract The discovery of the first cyclopropyl lactone-containing marine oxylipin in 1981 revealed a new class of compounds with interesting structural motifs exhibiting different bioactive properties. The development of stereodivergent approaches, structural elucidation strategies, and reassignment of certain configurations represented challenges for natural product researchers and built the fundament on a variety of publications. We herein will summarize the overall investigation on cyclopropyl lactone-containing marine oxylipins. Our focus lays thereby on the well-described constanolactones, solandelactones, and the related halicho- and neohalicholactone. This review embraces the isolation, proposed biosynthesis, characterization, and structural elucidation as well as summarizes investigated attractive (stereo-) selective approaches or methods toward common intermediates by structuring them according to the first applied retrosynthetic disconnection.

Wenn das Substrat sein Protein inhibiert

Hohe Konzentrationen an Acetaldehyd hemmen die Aldolase, das Enzym arbeitet nicht mehr. NMR-Spektroskopie, Rontgenstruktur und biochemische Analysen zeigen, wie es dazu kommt. Und sie offenbaren eine Losung: Wird eine Aminosaure ausgetauscht, ist das Enzym stabil und agiert so, wie es soll.

Corrigendum: Trading off stability against activity in extremophilic aldolases

Scientific Reports 6: Article number: 1790810.1038/srep17908; published online: January 19 2016; updated: November 11 2016

Elfte J-NOST-Konferenz in Indien

Im Dezember 2015 fand die elfte Konferenz des Junior-National Organic Symposium Trust (J-NOST XI) in Indien statt. Neben einer Delegation britischer Doktoranden reisten zum vierten Mal auch deutsche Doktoranden zu dieser Konferenz. Ermoglicht wurde dies durch Reisestipendien der GDCh (organisiert durch das Indigo-PhD-Netzwerk) und durch das internationale Promotionsprogramm der Universitat Regensburg (iPUR, fur Regensburger Doktoranden). Das National Institute of Science Education and Research (NISER) in Bhubaneswar im ostindischen Bundesstaat Odisha richtete die viertagige Konferenz aus.