Engineering 2‐oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by optimizing consecutive components

Abstract

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.

Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition

“…On reconstitution of the purified E2o proteins with substitutions identified with His298Asp-E1o and E3 into OGDHc, the formation of NADH from 2-OV could be detected in vitro (Table 3). Moreover, the formation of butyryl-CoA by the newly designed complexes could be detected by mass spectrometry [110]. Surprisingly, the identified His348 E2o variants retained the OGDHc activity toward the natural substrate 2-OG with the maximum reduction of catalytic efficiency (k cat /K m,2-OG ) by only 1.6-fold (His348Gln E2o).…”

“…The His348, conserved among known E2o sequences, is located near the proposed succinyl-binding site and was predicted to be responsible for recognition of the succinyl group (Figure 6) [66,99]. [110]. The succinyl-binding site contains residues Ser330 and Ser333 within hydrogenbonding distance of the carboxylate group of succinyldihydrolipoamide.…”

“…In the Farinas lab, SSM libraries were constructed for the E. coli E2o at positions Ser330, Ser333 and His348 were constructed [110]. The E2o variants with the His348Phe, His348Tyr, His348Gln and Ser333Met substitutions were identified from the screen.…”

“…Surprisingly, the identified His348 E2o variants retained the OGDHc activity toward the natural substrate 2-OG with the maximum reduction of catalytic efficiency (k cat /K m,2-OG ) by only 1.6-fold (His348Gln E2o). The E2o variants were also found to be active towards an even more hydrophobic substrate, 2-oxo-5-hexenoic acid (2-OHe), which contains a terminal alkene group that would be useful for further functionalization to increase product diversity (Figure 2, top) [110]. It needs to be noted that when assembled from unsubstituted E1o, E2o and E3 components, no NADH could be detected for OGDHc with 2-OV or 2-OHe as substrates.…”

Screen‐printing of nanocube‐based flexible microchips for the precise biosensing of ethanol during fermentation

“…For ethanol detection, the electrochemical biosensor mainly works by using alcohol oxidase (AOX) or alcohol dehydrogenase (ADH) as the biorecognizer to produce the response signal 26‐29 . Although AOX shows a higher catalytic activity to ethanol than ADH, it is poor in selectivity and can generally oxidize all short‐chain alcohols, such as methanol and butanol, which may also coexist with ethanol in some fermentation reactions 30,31 . Hence, ADH is more attractive for the fabrication of ethanol biosensors for fermentation applications.…”