Oxidative Decarboxylation of Short-Chain Fatty Acids to 1-Alkenes.

The enzymatic oxidative decarboxylation of linear short-chain fatty acids (C4:0-C9:0) employing the P450 monooxygenase OleT, O2 as the oxidant, and NAD(P)H as the electron donor gave the corresponding terminal C3 to C8  alkenes with product titers of up to 0.93 g L(-1) and TTNs of >2000. Key to this process was the construction of an efficient electron-transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite. This system allows for the biocatalytic production of industrially important 1-alkenes, such as propene and 1-octene, from renewable resources for the first time.

Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from Nitrosomonas europaea.

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g(parallel) = 2.24, A(parallel) = 18.4 mT and g(perpendicular) = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g(parallel) = 2.29, A(parallel) = 16.1 mT and g(perpendicular) = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu(2+) and three diamagnetic Cu(1+) per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per alphabetagamma-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe(3+) was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu(2+)-EPR signal.

A soluble form of ammonia monooxygenase in Nitrosomonas europaea.

Ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyzes the oxidation of ammonia to hydroxylamine. This study shows that AMO resides in the cytoplasm of the bacteria in addition to its location in the membrane and is distributed approximately equally in both subcellular fractions. AMO in both fractions catalyzes the oxidation of ammonia and binds [(14)C]acetylene, a mechanism-based inhibitor which specifically interacts with catalytically active AMO. Soluble AMO was purified 12-fold to electrophoretic homogeneity with a yield of 8%. AMO has a molecular mass of approximately 283 kDa with subunits of ca. 27 kDa (alpha-subunit, AmoA), ca. 42 kDa (beta-subunit, AmoB), and ca. 24 kDa (gamma-subunit, cytochrome c(1)) in an alpha(3)beta(3)gamma(3) sub-unit structure. Different from the beta-subunit of membrane-bound AMO, AmoB of soluble AMO possesses an N-terminal signal sequence. AMO contains Cu (9.4+/-0.6 mol per mol AMO), Fe (3.9+/-0.3 mol per mol AMO), and Zn (0.5 to 2.6 mol per mol AMO). Upon reduction the visible absorption spectrum of AMO reveals absorption bands characteristic of cytochrome c. Electron para-magnetic resonance spectroscopy of air-oxidized AMO at 50 K shows a paramagnetic signal originating from Cu(2+) and at 10 K a paramagnetic signal characteristic of heme-Fe.

Interaction of the mechanism-based inactivator acetylene with ammonia monooxygenase of Nitrosomonas europaea.

The ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyses the oxidation of ammonia to hydroxylamine. We have identified histidine 191 of AmoA as the binding site for the oxidized mechanism-based inactivator acetylene. Binding of acetylene changed the molecular mass of His-191 from 155.15 to 197.2 Da (+42.05), providing evidence that acetylene was oxidized to ketene (CH2CO; 42.04 Da) which binds specifically to His-191. It must be assumed that His-191 is part of the acetylene-activating site in AMO or at least directly neighbours this site.

Transcription of genes coding for metabolic key functions in Nitrosomonas europaea during aerobic and anaerobic growth.

Nitrosomonas europaea can grow under conditions of chemolithoautotrophic aerobic (oxygen as oxidant) as well as anaerobic [nitrogen dioxide (NO(2)) as oxidant] nitrification or chemoorganotrophic anaerobic pyruvate-dependent denitrification. In this study, the adaptation of the transcription (mRNA synthesis/concentration) of N. europaea to aerobic and anaerobic growth conditions was evaluated and the transcription of genes coding for metabolic key functions was analyzed: nitrogen and energy metabolism (amoA, hao, rh1, nirK, norB, nsc, aceE, ldhA, ppc, gltA, odhA, coxA), carbon dioxide fixation (cbbL), gluconeogenesis (ppsA), cell growth (ftsZ), and oxidative stress (sodB). During aerobic ammonia oxidation the specific activities of ammonia oxidation, nitrite reduction, and the growth rates correlated with the transcription level of the corresponding genes amoA/hao, nirK/norB/nsc, and cbbL/ftsZ. In anaerobically ammonia-oxidizing cells of N. europaea, the cellular mRNA concentrations of amoA, hao, rh1,coxA, cbbL, ftsZ, and sodB were reduced compared with aerobically nitrifying cells, but the mRNA levels of nirK, norB, and nsc were significantly increased. During anaerobic pyruvate-dependent denitrification, the mRNA abundance of nirK, norB, nsc, aceE, gltA, and odhA was increased, while the concentrations of amoA,hao, rh1, coxAcbbL, ftsZ, and sodB were significantly reduced. Temperature, pH value, and NH(4)(+), O(2), NO, and NO(2) concentrations had comparatively small effects on the transcription of the studied genes.

Functional and physiological evidence for a rhesus-type ammonia transporter in Nitrosomonas europaea.

Ammonium transporters form a conserved family of transport proteins and are widely distributed among all domains of life. The genome of Nitrosomonas europaea codes for a single gene (rh1) that belongs to the family of the AMT/Rh ammonium transporters. For the first time, this study provides functional and physiological evidence for a rhesus-type ammonia transporter in bacteria (N. europaea). The methylammonium (MA) transport activity of N. europaea correlated with the Rh1 expression. The K(m) value for the MA uptake of N. europaea was 1.8+/-0.2 mM (pH 7.25), and the uptake was competitively inhibited by ammonium [K(i)(NH(4) (+)) 0.3+/-0.1 mM at pH 7.25]. The MA uptake rate was pH dependent, indicating that the uncharged form of MA is transported by Rh1. An effect of the glutamine synthetase on the MA uptake was not observed. When expressed in Saccharomyces cerevisiae, the function of Rh1 from N. europaea as an ammonia/MA transporter was confirmed. The results suggest that Rh1 equilibrates the uncharged substrate species. A low pH value in the periplasmic space during ammonia oxidation seems to be responsible for the ammonium accumulation functioning as an acid NH(4) (+) trap.