The Escherichia coli 2‐oxoglutarate dehydrogenase complex ( OGDH c) comprises multiple copies of three enzymes—E1o, E2o, and E3—and transthioesterification takes place within the catalytic domain of E2o. The succinyl group from the thiol ester of S8‐succinyldihydrolipoyl‐E2o is transferred to the thiol group of coenzyme A (CoA), forming the all‐important succinyl‐CoA. Here, we report mechanistic studies of enzymatic transthioesterification on OGDH c. Evidence is provided for the importance of His375 and Asp374 in E2o for the succinyl transfer reaction. The magnitude of the rate acceleration provided by these residues (54‐fold from each with alanine substitution) suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid–base catalysis. Further evidence ruling out a role in acid–base catalysis is provided by site‐saturation mutagenesis studies at His375 (His375Trp substitution with little penalty) and substitutions to other potential hydrogen bond participants at Asp374. Taking into account that the rate constant for reductive succinylation of the E2o lipoyl domain ( LD o) by E1o and 2‐oxoglutarate (99 s −1 ) was approximately twofold larger than the rate constant for k cat of 48 s −1 for the overall reaction ( NADH production), it could be concluded that succinyl transfer to CoA and release of succinyl‐CoA, rather than reductive succinylation, is the rate‐limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2‐oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl‐CoA formation, so important for several biological processes including post‐translational succinylation of protein lysines. Enzymes 2‐oxoglutarate dehydrogenase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html ); dihydrolipoamide succinyltransferase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html ); dihydrolipoamide dehydrogenase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html ); pyruvate dehydrogenase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html ); dihydrolipoamide acetyltransferase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html ).

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Multienzyme complexes have the potential for green catalysis of sequential reactions. The Escherichia coli 2‐oxoglutarate dehydrogenase complex (OGDHc) was converted from a 2‐oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by engineering consecutive components. OGDHc catalyzes succinyl‐CoA synthesis in the Krebs cycle. OGDHc is composed of three components: E1o, 2‐oxoglutarate dehydrogenase; E2o, dihydrolipoylsuccinyl transferase; E3, dihydrolipoyl dehydrogenase. There are three substrate checkpoints. One is in E1o and two in E2o. OGDHc was reprogrammed to accept alternative substrates by evolving the E1o and E2o components. Wt‐ODGHc does not accept aliphatic substrates. E1o was previously engineered to accept a non‐natural aliphatic substrate, 2‐oxovalerate (2‐OV). E2o also required engineering to accept 2‐OV in the overall reaction. Hence, saturation mutagenesis libraries of E2o were screened for 2‐OV activity. E2o‐S333M, E2o‐H348F, E2o‐H348Q, and E2o‐H348Y were identified to show activity for 2‐OV in the reconstituted complex. Variants also displayed activity for larger aliphatic substrates.

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In this study, we introduce microwave-induced membrane distillation (MIMD) where microwave radiation is applied not only to heat water but also to enhance the biocidal effects of nanocarbon immobilized membranes. The three types of membranes used in this study were carbon nanotube immobilized membranes (CNIM), one functionalized with carboxylated nanotubes (CNIM-COOH), and graphene oxide immobilized membrane (GOIM). The membrane performances were evaluated based on the production of water vapor flux and the percentage cell growth inhibition due to the combined effect of microwaves and nanocarbon membranes. These combinations were most effective at a temperature of 80 and 60 °C for the removal of thermophilic and mesophilic cells, respectively. Under microwave heating, the CNIM exhibited the maximum biocidal effect (99.6% for thermophilic and 95.5% for mesophilic cells) followed by CNIM-COOH (92.3% for thermophilic and 65.8% for mesophilic cells) and GOIM (90.1% for thermophilic and 59.4% for mesophilic cells). They were all higher than a plain poly(tetrafluoroethylene) (PTFE) (82.3% for thermophilic and 41.6% for mesophilic cells) membrane without nanocarbons. In MIMD, the biocidal performance as well as the flux were improved due to thermal and nonthermal factors of microwave irradiation. The latter caused higher cell destruction due to the interaction of the microwave with the cellular matter, an improved water vapor flux (30−40%) due to localized superheating, and enhanced hydrogen bonding breakdown of water molecules. Furthermore, MIMD required much lesser (20−25%) energy than conventional MD to carry out the experiments under the same conditions.