Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+ to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+ were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+ to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm−2 and 0.255 mA cm−2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm−2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.
[…] Manually annotated and reviewed sequence data for 6PGDH were retrieved from the National Center for Biotechnology Information and Uniprot Database. Clustal Omega was used to perform a multiple sequence alignment. The sequence logo plot was created with the WebLogo 3.3 interface. […]
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