Jörg Pietruszka

Lights on and action! Controlling microbial gene expression by light

Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.

Spongistatins, Cynachyrolides, or Altohyrtins? Marine Macrolides in Cancer Therapy

Fascinating structures and stunning cytotoxicities characterize the altohyrtins A-C, the spongistatins 1-9, and cynachyrolide A. The first total syntheses reported by the groups from Evans and Kishi could unequivocally prove the structure of the extremely potent inhibitors of the tubulin polymerization.

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

Diastereo- and enantiomerically pure allylboronates: their synthesis and scope.

Allylboronates are highly attractive reagents for allyl additions. Enantiomerically pure, stable reagents with a stereogenic centre in alpha-position to boron are especially versatile, albeit often difficult to synthesize. Starting from boron-containing allyl alcohols 6 and 7, which are discussed in detail herein, a set of reagents were obtained via [3,3]-sigmatropic rearrangements and consecutive transformations in the side chain. The configurations could be established first by chemical correlation, but also by X-ray crystallography (16, 18, 34, and 39). Allyl additions were performed resulting in the formation of predominantly (Z)-configured homoallylic alcohols (31, 43-45) with high enantiomeric excess. Detailed investigations on the matched-mismatched interaction between the reagents 15/16 (and ent-15/ent-16, respectively) and isopropylidene glyceraldehyde 42d are presented.

Laccase‐catalyzed 3‐Arylation of 3‐Substituted Oxindoles

Enzymes are a powerful tool in organic synthesis, as the, often nontoxic, catalysts utilize mild reaction conditions, which can be applied and lead to exquisite stereoand regioselectivity in many transformations. Applications are not limited to the generation of basic chemicals, but enzymes have also been utilized in the syntheses of complex natural products. Laccases (EC 1.10.3.3), blue oxidoreductases of the multi-copper oxidase family, are capable of catalyzing the oxidation of diverse compounds, such as phenols, aminophenols, polyphenols, and related compounds. Recently, laccases have received considerable attention in organic synthesis, owing to their increased commercial availability, but especially because they are utilizing readily available aerial oxygen as oxidant for conversions under mild reaction conditions. In industry, laccases also find applications in various areas, such as food, paper, cosmetics, textile, and nanotechnologies. As part of our on-going endeavor to utilize enzymes in the synthesis of key-building blocks for active agent synthesis, we became interested in oxindoles. The oxindole core is a crucial structural motif present in a large number of naturally occurring and synthetic alkaloids exhibiting biological and pharmacological activities. Notable examples include those with anti-tumor and anti-cancer properties. Therefore, significant effort has been made to develop an efficient method for the synthesis of 3,3-disubstituted oxindole. The family of 3,3-diaryloxindoles is of special interest, since many of them are potent non-steroidal human antagonists or display anti-proliferative effects. f] Furthermore, 3-benzyl-3-phenyloxindole exists also as the core structure of an anti-cancer agent of Hoffmann–LaRoche. Despite the potential of the group of compounds, few methods for the synthesis of 3,3-diaryloxindoles or 3-benzyl-3phenyloxindoles have been described. Generally, unsymmetrical 3,3-diaryloxindoles can be prepared via two synthetic strategies. Recently, Sammakia et al. reported a versatile method for the synthesis of diaryloxindoles through palladium catalyzed a-arylation or nucleophilic aromatic substitution starting from 3-aryloxindoles and aryl halides. Alternatively, treatment of isatins with a Grignard reagent gives the 3-aryl-3-hydroxyloxindoles, which are subsequently subjected to a trifluoroacetic acid-mediated Friedel–Crafts alkylation to afford the unsymmetrical diaryloxindoles (Scheme 1). It is worth noting that these Friedel–Crafts-reactions proceed with ortho-selectivity. Aiming at an arylation with different regioselectivity, we envisaged a laccase-catalyzed arylation of 3-substituted oxindoles by catechols. The process is assumed to be initiated by a laccase-catalyzed oxidation of the catechol A to the corresponding o-quinone C which subsequently undergoes a Michael addition with the oxindole B as nucleophile, forming the unsymmetrical aryloxindole D as product with para/meta selectivity (Scheme 2).

Gas chromatographic enantioseparation of allenes

Abstract Enantioselective gas chromatography with modified cyclodextrins as stationary phases is successfully used to separate chiral allenic hydrocarbons - including kinetically labile 1,2-cyclooctadiene - and 1-halo-1,2-butadienes.

Latex clearing protein-an oxygenase cleaving poly(cis-1,4-isoprene) rubber at the cis double bonds.

ABSTRACT Gordonia polyisoprenivorans strain VH2, a potent rubber-degrading actinomycete, harbors two latex clearing proteins (Lcps), which are known to be essential for the microbial degradation of rubber. However, biochemical information on the exact role of this protein in the degradation of polyisoprene was lacking. In this study, the gene encoding Lcp1VH2 was heterologously expressed in strains of Escherichia coli, the corresponding protein was purified, and its role in rubber degradation was examined by measurement of oxygen consumption as well as by chromatographic and spectroscopic methods. It turned out that active Lcp1VH2 is a monomer and is responsible for the oxidative cleavage of poly(cis-1,4-isoprene) in synthetic as well as in natural rubber by the addition of oxygen (O2) to the cis double bonds. The resulting oligomers possess repetitive isoprene units with aldehyde (CHO-CH2—) and ketone (—CH2-CO-CH3) functional groups at the termini. Two fractions with average isoprene contents of 18 and 10, respectively, were isolated, thus indicating an endocleavage mechanism. The activity of Lcp1VH2 was determined by applying a polarographic assay. Alkenes, acyclic terpenes, or other rubber-like polymers, such as poly(cis-1,4-butadiene) or poly(trans-1,4-isoprene), are not oxidatively cleaved by Lcp1VH2. The pH and temperature optima of the enzyme are at pH 7 and 30°C, respectively. Furthermore, it was demonstrated that active Lcp1VH2 is a Cu(II)-containing oxygenase that exhibits a conserved domain of unknown function which cannot be detected in any other hitherto-characterized enzyme. The results presented here indicate that this domain might represent a new protein family of oxygenases.

Stereoselective synthesis of both enantiomers of rugulactone.

The stereoselective total synthesis of both enantiomers of rugulactone 1 has been completed by applying enantioselective allyl additions as key steps. Two different strategies based on highly stable and enantiomerically pure α-substituted allylboronic esters 2 and 3 were performed starting from boronic ester 4.

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.

A Diastereoselective One-Pot, Three-Step Cascade toward α-Substituted Allylboronic Esters

A new highly diastereoselective synthesis of chiral α-substituted allylboronic esters, based on a one-pot, three-step cascade, is presented. The palladium- and acid-cocatalyzed reaction cascade involves a desilylation of a TBS-protected allylic alcohol, borylation, and addition of an allyl group to an aldehyde. Herein we present the first application of a TBS-protected allylic alcohol in a palladium-catalyzed borylation/allylation reaction.