Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80 °C and pH 6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures

Increased postprandial glycaemia, insulinemia, and lipidemia after 10 weeks’ sucrose-rich diet compared to an artificially sweetened diet: a randomised controlled trial

The importance of exchanging sucrose for artificial sweeteners on risk factors for developing diabetes and cardiovascular diseases is not yet clear. Objective: To investigate the effects of a diet high in sucrose versus a diet high in artificial sweeteners on fasting and postprandial metabolic profiles after 10 weeks.Healthy overweight subjects were randomised to consume drinks and foods sweetened with either sucrose (∼2 g/kg body weight) (n = 12) or artificial sweeteners (n = 11) as supplements to their usual diet. Supplements were similar on the two diets and consisted of beverages (∼80 weight%) and solid foods (yoghurts, marmalade, ice cream, stewed fruits). The rest of the diet was free of choice and ad libitum. Before (week 0) and after the intervention (week 10) fasting blood samples were drawn and in week 10, postprandial blood was sampled during an 8-hour meal test (breakfast and lunch).After 10 weeks postprandial glucose, insulin, lactate, triglyceride, leptin, glucagon, and GLP-1 were all significantly higher in the sucrose compared with the sweetener group. After adjusting for differences in body weight changes and fasting values (week 10), postprandial glucose, lactate, insulin, GIP, and GLP-1 were significantly higher and after further adjusting for differences in energy and sucrose intake, postprandial lactate, insulin, GIP, and GLP-1 levels were still significantly higher on the sucrose-rich diet.A sucrose-rich diet consumed for 10 weeks resulted in significant elevations of postprandial glycaemia, insulinemia, and lipidemia compared to a diet rich in artificial sweeteners in slightly overweight healthy subjects

Aktivierung von H2 und CO2 : Einblicke in biologische homo- und bimetalische Katalyse unter Anwendung von Schwingungsspektroskopie

The activation of small molecules, such as hydrogen or carbon dioxide, is an important part in a renewable, green chemistry in the future in our society. In this context, nature already shaped metalloenzymes, which are capable of catalyzing a wide variety of reactions. In order to exploit their biotechnological potential and provide an optimum performance, a fundamental understanding of their catalytic mechanisms is inevitable. This thesis presents an interdisciplinary approach providing detailed insights into the biochemical and spectroscopic properties of a soluble hydrogenase (SH) from Hydrogenophilus thermoluteolus TH-1T (Ht). Additionally, a formate dehydrogenase (FDH) from Rhodobacter capsulatus (Rc) was investigated by means of vibrational spectroscopy. The first part of this thesis discusses kinetic and activity assays of the HtSH. Complementary, infrared (IR) absorption spectroscopy in combination with electron paramagnetic resonance (EPR) spectroscopy was carried out on the same enzyme samples in order to gain precise information about redox species of the catalytic center before and after activation as well as enzymatic integrity. The HtSH exhibits catalytic H2-mediated Nicotinamide adenine dinucleotide (NAD+) reduction in the presence of O2 at elevated temperatures displaying an optimum at 50 °C. The IR spectroscopic characterization reveals a rich set of different oxidized and reduced intermediates. Notably, in the oxidized state an unprecedented upshifted CO stretching mode is observed at 1993 cm-1. Within the reduced species, the Nia-C signal in the EPR spectrum of H2-treated sample was mainly found at temperatures below 10 K, which could presumably be a result of fast spin-lattice relaxation related to magnetic coupling with another cofactor that is paramagnetic under these reducing conditions. In conclusion, this might serve as an explanation why Nia-C and other paramagnetic active site species often are not detected. Furthermore, to explain the molecular details of the O2-tolerance origin in soluble [NiFe]-hydrogenases, a bioinspired heterobimetallic S-oxygenated [NiFe] complex was synthesized. This complex was fully analyzed by vibrational spectroscopy in comparison to the oxygen-free reference species. Using further experimental and computational techniques, the electronic structures were elucidated with emphasis on the bridging sulfenato moiety. Based on the obtained results, novel strategies of exploring S-oxygenated intermediatesin hydrogenases are proposed. The last part of this thesis is dedicated to the investigation of a FDH. Here, resonance Raman (RR) spectroscopy in combination with computational methods were applied. The research focuses primarily on the first coordination sphere of the Molybdenum ion of the active site in the oxidized and reduced state. Spectra of different structural models were calculated and compared with the experimental data. Based on this multidisciplinary approach, a mixture of reduced intermediate states were assigned and compared to the existing proposed catalytic cycles. Additionally, the binding mode of the inhibitor molecule azide was investigated by IR spectroscopy. The direct binding mechanism to the Molybdenum ion proposed so far cannot be verified by the obtained data. The presented results demonstrate that a binding in the proximity of the active site is rather likely.Die Aktivierung von kleinen Molekülen, wie beispielsweise Wasserstoff oder Kohlenstoffdioxid, ist ein entscheidener Faktor im Rahmen in einer zukünftigen, erneuerbaren und grünen Chemie. Die Natur hat dafür Metalloenzyme entwickelt, die in der Lage sind, eine große Anzahl verschiedener Reaktionen zu katalysieren. Um deren biotechnologischen Potentiale möglichst vollständig auszunutzen und eine optimale Leistungsfähigkeit zu entwickeln, ist ein fundamentales Verständnis der zugrundeliegenden katalytischen Mechanismen unumgänglich. Diese Doktorarbeit präsentiert einen interdisziplinären Ansatz, um umfassende Einblicke in die biochemischen und spektroskopischen Eigenschaften einer löslichen Hydrogenase (SH) aus Hydrogenophilus thermoluteolus TH-1T (Ht) und einer Formiat Dehydrogenase (FDH) aus Rhodobacter capsulatus (Rc) zu gewinnen.Im ersten Teil der vorliegenden Arbeit werden kinetische sowie Aktivitätsversuche zur HtSH diskutiert. Zusätzlich, werden mittels Infrarot- (IR) sowie Elektronenspinresonanzspektroskopie (EPR) weitere wichtige Informationen zu unterschiedlichen Spezien des aktiven Zentrums und zur enzymatischen Integrität generiert. Die HtSH zeigt katalytische, wasserstoffabhänige Nicotinamidadenindinukleotid- (NAD+) Reduktion. Dies geschieht in der Gegenwart von O2 bei erhöhten Temperaturen mit einem Optimum von 50 °C. Mittels IR Spektroskopie kann eine Reihe von oxidierten und reduzierten Intermediaten charakterisiert werden. Hierbei ist bemerkenswert, dass im oxidierten Zustand eine bisher noch nicht entdeckte, hochverschobene CO Streckschwingung beobachtet wird. In den reduzierten Zuständen wird im EPR Spektrum das Nia-C Signal, der mit Wasserstoff behandelten Probe, nur bei Temperaturen unterhalb von 10 K detektiert. Dies könnte das Resultat einer schnellen Spin-Gitter-Relaxierung mit einem benachbarten Kofaktor sein, der unter diesen Bedingungen ebenfalls reduziert vorliegt. Das wäre eine mögliche Erklärung, warum Nia-C und andere paramagnetische Zustände des aktiven Zentrums häufig nichtbeobachtet werden. Um das Verständnis der molekularen Details zur Sauerstofftoleranz von löslichen Hydrogenasen voranzutreiben, wurde ein bioinspirierter S-oxygenierter [NiFe] Komplex vollständig mittels Schwingungsspektroskopie charakterisiert. Durch die Anwendung von weiteren experimentellen und theoretischen Techniken konnten die elektronischen Strukturen des Komplexes und seiner sauerstofffreien Referenzstruktur aufgeklärt werden. Hierbei lag ein besonderer Schwerpunkt auf der verbrückenden Sulfenatoeinheit. Anhand der vorliegenden Ergebnisse können neuartige Strategien vorgeschlagen werden, um S-oxygenierte Intermediate in Hydrogenasen zu untersuchen. Der letzte Teil der Arbeit beschäftigt sich mit der Untersuchung einer FDH. Hierbei wurde Resonanz-Raman-Spektroskopie (RR) in Kombination mit computergestützten Modellrechungen verwendet. Die Untersuchung fokusierte sich vor allem auf die erste Koordinationsspähre des Molybdenumions des aktiven Zentrums im oxidierten und reduzierten Zustand. Unterschiedliche, berechnete Spektren wurden mit den experimentellen Daten verglichen. Auf diesem Ansatz basierend konnten verschiedene reduzierte Intermediate zugeordnet und mit den bisher bestehenden katalytischen Zyklen verglichen werden. Zusätzlich wurde noch die Bindung des Inhibitors Azid mittels IR Spektroskopie überprüft. Die so gewonnenen Daten demonstrieren, dass ein direkter Bindungsmechanismus an das Molybdänion, wie es kürzlich vorgeschlagen wurde, eher unwahrscheinlich gegenübereiner möglichen Bindung in der Nähe des aktiven Zentrums ist

Reagent for the Enzymatic Determination of Serum Total Triglycerides with Improved Lipolytic Efficiency

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