ADHs Cthe_0394, Cthe_0101, and Cthe_2579 were expressed at 78%, 2

ADHs Cthe_0394, Cthe_0101, and Cthe_2579 were expressed at 78%, 24%, and 9% of the levels of AdhE, respectively, suggesting that they may also be involved in formation of ethanol from acetaldehyde, albeit at lower levels. Two other zinc-containing ADH GroES-like heat shock proteins, Cthe_0388 and Cthe_2445, were also detected, the former being more highly expressed ( Additional file 4). While crude

cell-free extract enzyme activities have shown the presence of both NADH and NADPH-dependent ADH activities, sequence analysis could not verify the substrate specificities of these enzymes. Acetyl-CoA can be converted into acetate directly via acetate thiokinase (ATK) or indirectly through an acetyl phosphate intermediate using contiguously encoded phosphotransacetylase (PTA) and acetate kinase (ACK). While activities of PTA (Cthe_1029) and ACK (Cthe_1028) have been verified in C. thermocellum[50], selleck inhibitor and ACK has been purified and characterized [86], the substrate specificity of the putative ATK (Cthe_0551) has not been determined. Although both reactions generate ATP, ATK does so using AMP and PPi, whereas PTA and ACK use ADP and Pi. This in turn has an impact on the thermodynamics of each reaction. The free energy of acetate production

using PTA and ACK is more thermodynamically favourable than using ATK (ΔG˚’ = −4 kJ mol-1 vs +9 kJ mol-1), filipin and thus PTA and ACK are proposed to favour acetate production from acetyl-CoA, while ATK favours acetyl-CoA production from acetate. While Raman et al. report low mRNA levels of pta and ack and higher levels of atk[37], 2D-HPLC-MS/MS showed that all three proteins were detected

at comparable levels (Figure  3b). Expression of all three enzymes remained constant throughout fermentation. H2 generation pathways The genome of C. thermocellum encodes four putative hydrogenases (H2ases), including an energy conserving Ech-like Fd-dependent [NiFe]-H2ase (Cthe_3019-3024) and 3 Fe-only H2ase catalytic subunits (Cthe_0342, Cthe_0430, Cthe_3003). Transcription of all of these subunits has been confirmed using RT-PCR [22]. Enzyme assays have shown that NADPH-dependent H2ase activity is 5 to 10-fold higher than Fd and NADH-dependant H2ase activities [4, 55]. The presence of a gene similar to the NADPH-binding subunit of glutamate synthase (Cthe_3004) adjacent to Cthe_3003 suggests that it may form a dimer with Cthe_3003 capable of generating NADPH from H2[18]. 2D-HPLC-MS/MS reveals that both subunits are highly expressed, while subunits comprising both Fd-dependent [NiFe]-H2ase and Rhodobacter nitrogen fixation (RNF)-like NADH:Fd oxidoreductase were detected in low amounts or not at all (Figure  3c), consistent with enzyme activity profiles [4, 55] and mRNA profiles [37]. This leads to the question of how reduced Fd, formed by PFO, is reoxidized.

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