03 Fakultät Chemie
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Item Open Access Functional role of lanthanides in enzymatic activity and transcriptional regulation of Pyrroloquinoline quinone-dependent alcohol dehydrogenases in Pseudomonas putida KT2440(2017) Wehrmann, Matthias; Billard, Patrick; Martin-Meriadec, Audrey; Zegeye, Asfaw; Klebensberger, JanoschThe oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca2+-dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La3+, Ce3+, Pr3+, Sm3+, or Nd3+. Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.Item Open Access Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole-cell screening approach(2018) Wehrmann, Matthias; Klebensberger, JanoschDue to their ability for direct electron transfer to electrodes, the utilization of rare earth metals as cofactor, and their periplasmic localization, pyrroloquinoline quinone‐dependent alcohol dehydrogenases (PQQ‐ADHs) represent an interesting class of biocatalysts for various biotechnological applications. For most biocatalysts protein stability is crucial, either to increase the performance of the protein under a given process condition or to maximize robustness of the protein towards mutational manipulations, which are often needed to enhance or introduce a functionality of interest. In this study, we describe a whole‐cell screening assay, suitable for probing PQQ‐ADH activities in Escherichia coli BL21(DE3) cells, and use this assay to screen smart mutant libraries for increased thermal stability of the PQQ‐ADH PedE (PP_2674) from Pseudomonas putida KT2440. Upon three consecutive rounds of screening, we identified three different amino acid positions, which significantly improve enzyme stability. The subsequent combination of the beneficial mutations finally results in the triple mutant R91D/E408P/N410K, which not only exhibits a 7°C increase in thermal stability but also a twofold increase in residual activity upon incubation with up to 50% dimethyl sulfoxide (DMSO), while showing no significant difference in enzymatic efficiency (kcat/KM).Item Open Access SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions(2016) Colley, Brendan; Dederer, Verena; Carnell, Michael; Kjelleberg, Staffan; Rice, Scott A.; Klebensberger, JanoschPseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions.Item Open Access Durch Überexpression in der Hefe Pichia pastoris zu erhöhter Enantioselektivität : ein neues Kapitel in der Anwendung von Schweineleberesterase(2001) Musidlowska, Anna; Lange, Stefan; Bornscheuer, Uwe TheoLipases and Esterases can be used as efficient biocatalysts for the preparation of a wide variety of optically pure compounds. Whereas a range of lipases - especially of microbial origin - are commercially available, only a few esterases can be obtained for the kinetic resolution of racemates or desymmetrization. In the majority of publications, pig liver esterase (PLE) is used, which is isolated from pig liver by extraction. Although it could be demonstrated, that this preparation can convert a broad range of compounds at partially very high stereoselectivity, its application is encountered with a number of disadvantages.Item Open Access Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis(2004) Budde, Michael; Maurer, Steffen Christian; Schmid, Rolf D.; Urlacher, Vlada B.The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelat affinity chromatography (IMAC) and characterised. CYP102A2 is a 119 kDa self-sufficient monooxygenase, consisting of an FMN/FAD–containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from Bacillus megaterium (59%) and CYP102A3 from Bacillus subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 450 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ù-3, ù-2 and ù-1 hydroxylated fatty acids. Activity was highest towards oleic and linoleic acid (KM=17.4 ± 1.4 ìM, kcat= 2244 ± 72 min-1), linoleic acid (KM=12.25 ± 1.8 ìM, kcat= 1950 ± 84 min-1). Comparison of CYP102A2 homology model to CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in catalytic properties of these two enzymes.