Markus Dick

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.

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.

Probing the acetaldehyde-sensitivity of 2-deoxy-ribose-5-phosphate aldolase (DERA) leads to resistant variants

The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a synthetically attractive enzyme because of its ability to perform CC-couplings stereoselectively, the enzyme uses acetaldehyde as nucleophile and thus produces true aldols rather than ketols, and may add two acetaldehyde molecules onto one electrophile. However, DERA produces crotonaldehyde as side reaction from acetaldehyde which is then an irreversible inhibitor forming a covalent Michael-adduct within the active site in particular with cysteine 47 (Dick et al., 2016). This inhibition can be resolved by mutating C47 to non-nucleophile amino acids. Still, the inhibition is not an on-off-feature and the present mutagenesis study illustrates that there must be a C47-independent inactivation mechanism. As a practical result: The virtually fully resistant mutant C47L was found, which shows no loss in stereoselectivity, - this renders this variant as promising catalyst.

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein–protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 – the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.

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.

Pyrazolidine‐3,5‐dione‐based inhibitors of phosphoenolpyruvate carboxylase as a new class of potential C4 plant herbicides

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway of many of the worlds worst weeds and a valuable target to develop C4 plant‐selective herbicides. By virtual screening, analog synthesis, and in vitro validation, we identified pyrazolidine‐3,5‐diones as a new class of small molecules with inhibitory potential down to the submicromolar range against C4 PEPC and a selectivity factor of up to 16 over C3 PEPC. No other biological activity has yet been reported for the best compound, (3‐bromophenyl)‐4‐(3‐hydroxybenzylidene)‐pyrazolidine‐3,5‐dione. A systematic variation in the substituents allowed the derivation of a qualitative structure–activity relationship. These findings make this compound class highly interesting for further investigations toward generating potent, C4 plant‐selective herbicides with a low potential for unwanted effects.

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