Chen, HuiZhu, ZhiguangHuang, RuiZhang, Y. H. Percival2017-01-182017-01-182016-11-022045-2322http://hdl.handle.net/10919/74379Engineering 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 x 6.4 x 10(4)-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), similar to 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 degrees 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.? - ? (8) page(s)application/pdfenCreative Commons Attribution 4.0 Internationalketol-acid reductoisomeraseenzymatic biofuel cellsalcohol-dehydrogenaseescherichia-colicofactorspecificityglucosedesignnadhbiocatalysisArticle - Refereedhttps://doi.org/10.1038/srep36311627805055