Catabolic enzymes in the fumonisin degradation pathway of Sphingopyxis sp. MTA144

Fumonisine sind krebserregende Mycotoxine, die von Fusarium verticillioides und anderen phytopathogenen Pilzen produziert werden. Sie sind weltweit vor allem auf Mais zu finden und verursachen schwere Erkrankungen bei Mensch und Tier. Da chemische und physikalische Dekontaminationsmethoden Fumonisine nicht ausreichend inaktivieren können, ist unsere Strategie die biologische Entgiftung des Mykotoxins durch enzymatische Inaktivierung im Verdauungstrakt von Tieren. Wir identifizierten in einem alpha-Proteobakterium, Sphingopyxis sp. MTA144, einen Gencluster, der mit dem Fumonisinabbau in Verbindung steht. Die darin enthaltenen elf „open reading frames“ inkludierten Transkriptionsregulatoren, Transmembranproteine und katabolische Enzyme. Wir konnten die Funktion von vier Enzymen identifizieren. Der Abbauweg beginnt mit der Esterhydrolyse des FB1 durch die Carboxylesterase FumD, wodurch hydrolysiertes FB1 (HFB1) entstand, das entweder durch die Aminotransferase FumI deaminiert oder durch die Alkoholdehydrogenase FumH oxidiert wird. Die Reaktionsprodukte wurden als 2-keto-HFB1 bzw. 15-keto-HFB1 identifiziert. Das Reaktionsprodukt von beiden Enzymen, 2,15-diketo-HFB1, dient wiederum der Acetolactatsynthase FumK als bevorzugtes Substrat. Biochemische und enzymkinetische Parameter wurden für die Aminotransferase FumI bestimmt: Das bevorzugte Kosubstrat für FumI war Pyruvat. Das pH Optimum lag bei pH 8,5, das Temperaturoptimum bei 35C. Nach einstündiger Inkubation bei 60C war das Enzym vollständig inaktiviert. Restaktivität konnte nach 1 h Inkubation bei pH 4,0 oder mehr gemessen werden. Der KM Wert lag in Gegenwart von 5 mM Pyruvat bei 1.1 M, die spezifische Aktivität bei 2.2 mol/min/mg. Diese Arbeit klärte den Abbauweg von Fumonisin in Sphingopyxis sp. MTA144 auf und lieferte Enzyme für eine zukünftige Anwendung zu Entgiftung des Mykotoxins im Verdauungstrakt von Tieren.Fumonisins are mycotoxins predominantly produced by the plant pathogen Fusarium verticillioides and can be found worldwide especially in maize. Fumonisin B1 (FB1), the most abundant representative, is carcinogenic in rodents, causes porcine pulmonary edema and equine leukoencephalomalacia in livestock and human exposure to FB1 is linked to esophageal cancer and neural tube defects. FB1 is resistant to detoxification by chemical and physical methods. Therefore we are working on a biological detoxification strategy, based on enzymatic inactivation of fumonisins in the gastrointestinal tract of animals. We identified a gene cluster in Sphingopyxis sp. MTA144 which enables this alpha-proteobacterium to degrade fumonisins. Eleven open reading frames were predicted to encode proteins involved in the degradation pathway, including transcriptional regulators, transmembrane proteins and catabolic enzymes. We experimentally confirmed the function of four catabolic enzymes by heterologous gene expression followed by determination of enzyme activities. The carboxylesterase FumD catalyzed the hydrolysis of FB1. As a result, hydrolyzed FB1 (HFB1) was formed, which could either be deaminated by the aminotransferase FumI in presence of pyruvate and pyridoxal phosphate or dehydrated by the alcohol dehydrogenase FumH in presence of ß-Nicotinamide adenine dinucleotide. The resulting products of these reactions were 2-keto-HFB1 and 15-keto-HFB1, respectively. The reaction product of both enzymes, 2,15-diketo-HFB1, was the preferred substrate for the acetolactate synthase FumK. Kinetic parameters and enzyme characteristics were determined for FumI to evaluate its suitability as a feed enzyme. The preferred co-substrate of FumI was pyruvate. The enzyme was active in the range of pH 6.5 9.7 with an optimum at pH 8.5 and in a broad temperature range between 6C and 50C with an optimum at 35C. FumI was completely inactivated after 1 h incubation at 60C. Residual activity could be determined after preincubation for 1 h at pH 4.0 or higher. The KM value 1.1 M was determined with the 5 mM pyruvate. Specific activity was 2.2 mol/min/mg. In conclusion, this work elucidated the fumonisin degradation pathway of Sphingopyxis sp. MTA144 and provided enzymes for possible further application as a feed additive for gastrointestinal fumonisin detoxification.Doris HartingerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Univ. für Bodenkultur, Diss., 2011OeBB(VLID)193062

Enhancement of solubility in <it>Escherichia coli </it>and purification of an aminotransferase from <it>Sphingopyxis </it>sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Abstract Background Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. Results When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG) used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3). Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3), which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the binding buffer were performed to obtain 1.45 mg of apparently homogeneous aminotransferase per liter of expression culture. Conclusions We found that only reduction of temperature, but not reduction of expression level or fusion to maltose-binding protein helped to produce correctly folded, active aminotransferase FumI in E. coli. Our results may provide a starting point for soluble expression of related aminotransferases or other aggregation-prone proteins in E. coli.</p

The Effect of Uni- and Bilateral Thalamic Deep Brain Stimulation on Speech in Patients With Essential Tremor: Acoustics and Intelligibility

BackgroundDeep brain stimulation (DBS) of the ventral intermediate nucleus (VIM) is performed to suppress medically-resistant essential tremor (ET). However, stimulation induced dysarthria (SID) is a common side effect, limiting the extent to which tremor can be suppressed. To date, the exact pathogenesis of SID in VIM-DBS treated ET patients is unknown. ObjectiveWe investigate the effect of inactivated, uni- and bilateral VIM-DBS on speech production in patients with ET. We employ acoustic measures, tempo, and intelligibility ratings and patient's self-estimated speech to quantify SID, with a focus on comparing bilateral to unilateral stimulation effects and the effect of electrode position on speech. MethodsSixteen German ET patients participated in this study. Each patient was acoustically recorded with DBS-off, unilateral-right-hemispheric-DBS-on, unilateral-left-hemispheric-DBS-on, and bilateral-DBS-on during an oral diadochokinesis task and a read German standard text. To capture the extent of speech impairment, we measured syllable duration and intensity ratio during the DDK task. Naive listeners rated speech tempo and speech intelligibility of the read text on a 5-point-scale. Patients had to rate their ability to speak. ResultsWe found an effect of bilateral compared to unilateral and inactivated stimulation on syllable durations and intensity ratio, as well as on external intelligibility ratings and patients' VAS scores. Additionally, VAS scores are associated with more laterally located active contacts. For speech ratings, we found an effect of syllable duration such that tempo and intelligibility was rated worse for speakers exhibiting greater syllable durations. ConclusionOur data confirms that SID is more pronounced under bilateral compared to unilateral stimulation. Laterally located electrodes are associated with more severe SID according to patient's self-ratings. We can confirm the relation between diadochokinetic rate and SID in that listener's tempo and intelligibility ratings can be predicted by measured syllable durations from DDK tasks

The effect of uni- and bilateral thalamic Deep Brain Stimulation on speech in patients with Essential Tremor: production and perception

Titel im Abstract: The effect of uni- and bilateral thalamic deep brain stimulation on speech in patients with essential tremor: Acoustics and intelligibilit