A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

A Hitchhiker’s Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

“…sTHs are related to the disulfide reductase family of flavoproteins, and retain an exceptional CXXXXT domain (instead of the typical CXXXXC domain for reducing disulfides) which promotes tight FAD-embedding to the active site (Figure C, right panel) and allows for efficient transhydrogenation . As reported for several flavoproteins, SthA also exhibits oxidase activity both in the absence of an oxidized cofactor as acceptor and during transhydrogenation.…”

“…The purified SthA catalyzes the concomitant oxidation of NADPH and reduction of the non-natural substrate thio-NAD + with a turnover number between 170 and 260 s –1 and K M values around one hundred micromolar for both substrates . The SthA-mediated reaction consuming NADH while reducing thioNADP + has also been recently characterized, revealing a lower k cat value of 10–15 s –1 , and higher affinity for thioNADP + compared to NADH (0.1 and 2.6 mM, respectively) . Substrate inhibition occurs in both directions of reaction with NADPH, NADH and thioNADP + , as well as high concentrations of phosphate ion decrease the activity of SthA to 15–25% compared to a phosphate-free reaction medium.…”

“… 133 , 134 In E. coli , SthA adopts a globular conformation characterized by 8 protomers with an overall radius around 8 nm. 135 High resolution structures of the octameric complex have not yet been resolved for any sTH homologue, but the predicted monomer structure ( Figure 8 C, left panel) is available on the AlphaFold database.…”

“… 136 The SthA-mediated reaction consuming NADH while reducing thioNADP + has also been recently characterized, revealing a lower k cat value of 10–15 s –1 , and higher affinity for thioNADP + compared to NADH (0.1 and 2.6 mM, respectively). 135 Substrate inhibition occurs in both directions of reaction with NADPH, NADH and thioNADP + , as well as high concentrations of phosphate ion decrease the activity of SthA to 15–25% compared to a phosphate-free reaction medium. The enzyme works optimally at pH 7.5–8.5 and at a temperature of 35 °C, maintaining full activity for at least 3 weeks when incubated at 4 °C.…”

“…sTHs are related to the disulfide reductase family of flavoproteins, and retain an exceptional CXXXXT domain (instead of the typical CXXXXC domain for reducing disulfides) which promotes tight FAD-embedding to the active site (Figure 8C, right panel) and allows for efficient transhydrogenation. 135 SthA also exhibits oxidase activity both in the absence of an oxidized cofactor as acceptor and during transhydrogenation. This reactivity with dioxygen leads to a marginal formation of hydrogen peroxide and superoxide anion (together they intercept 2% of reducing equivalents from NADH to thioNADP + , while the remaining 98% is used for transhydrogenation).…”