Enzymes of two clostridial amino-acid fermentation pathways
Two enzymes of the alanine fermentation pathway of Clostridium propionicum were purified and biochemically characterized. The enzyme (R)-lactyl-CoA dehydratase catalyzing the difficult dehydration reaction of (R)-lactyl-CoA to acrylyl-CoA could be partially purified under strict anaerobic conditions...
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|Summary:||Two enzymes of the alanine fermentation pathway of Clostridium propionicum were purified and biochemically characterized. The enzyme (R)-lactyl-CoA dehydratase catalyzing the difficult dehydration reaction of (R)-lactyl-CoA to acrylyl-CoA could be partially purified under strict anaerobic conditions. Green fractions of component D and dark brown fractions of component A (activator) were obtained in separate enzyme pools. Lactyl-CoA was identified in MALDI mass spectrometry (m/z = 840) as product of the hydration reaction after assaying lactyl-CoA dehydratase activity by mixing component D and component A in presence of acrylyl-CoA, ATP, Mg+2 and dithionite. A second enzyme of the alanine fermentation pathway was identified catalyzing the ammonification of acrylyl-CoA in the same micro-organism. Beta-Alanyl-CoA ammonia-lyase activity is 300-fold increased in cell-free extracts of beta-alanine grown cells (143 U mg-1) as compared to their D,L-alanine counterparts (0.44 U mg-1). Therefore the enzyme was readily purified from beta-alanine grown cells and a high final specific activity (1033 U mg-1) was found after one Source 15-Q anion exchange column. The enzyme has a molecular size of 95 kDa and is composed of six 16 kDa subunits (alpha6). It shows high catalytic activity towards acrylyl-CoA (Km = 23 µM) independently of the ammonia concentration (Km = 70 mM) at almost limiting diffusion rate conditions (kcat/Km = 107 M-1 s-1). In the reverse reaction the elimination of ammonia (beta-alanyl-CoA Km = 210 µM) is apparently 70 % inhibited at 100 mM NH4Cl while the Km for beta-alanyl-CoA remains unchanged.
In the glutamate fermentation pathway via 3-methylaspartate, the enzyme catalyzing the reduction of crotonyl-CoA to butyryl-CoA was purified from Clostridium tetanomorphum in a complex with the electron transfer flavoprotein (ETF). Butyryl-CoA dehydrogenase-ETF was characterized as a 360 kDa alpha,beta,gamma-heteromer composed of three different subunits: the 40 kDa alpha-subunit (butyryl-CoA dehydrogenase) the 36 kDa beta-subunit (ETF alpha-subunit) and the 28 kDa gamma-subunit (ETF beta-subunit). Flavin content was less than 1 mol FAD but the enzyme could be reconstituted with additional FAD (2 mol) after one hour incubation at saturation conditions. The purified BCD-ETF complex presents butyryl-CoA dehydrogenase activity with ferricenium as electron acceptor (1 U mg-1) and diaphorase activity in both oxic and anoxic atmospheres. Crotonyl-CoA reduction (20 U mg-1) could be measured anoxically at 340 nm only in the presence of additional FAD (50 µM) and the concomitant diaphorase activity was approximately 1 U mg-1. In situ localization using immuno- and electron microscopy techniques revealed that the complex is evenly distributed over the cytoplasma of the bacteria. This result excludes a direct involvement of the exergonic crotonyl-CoA reduction in energy conservation via an electrochemical ion gradient. Further it paved the way to the discovery of a third mode of energy conservation|
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