Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries.

Hui Chen,Rui Huang,Yi-Heng Percival Zhang,Zhiguang Zhu

doi:10.1038/srep36311

Abstract

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.

Highlights

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+
The alignment of the loop region shows that three amino acids in NADP+-preferred 6PGDHs are highly conservative (Fig. 1b), which are asparagine, arginine and threonine (Asn[32], Arg[33] and Thr34) in Tm6PGDH
According to the above information, we hypothesized that the changes in Asn[32], Arg[33], and Thr[34] could enable Tm6PGDH to work on NAD+

Summary

Results

Amino acid-sequence alignment and structure analysis of 6PGDH. We analyzed the Rossmann fold domain of two kinds of 6PGDHs based on their coenzyme specificity (Fig. 1a). The mutant N32D has the replacement of the alkaline asparagine with the acidic aspartate, deleting the former hydrogen bond between the 2′-phosphate of NADP+ and the asparagine residue of the wide type Tm6PGDH (Fig. 2a) and resulting in a decrease in the binding affinity of the mutant N32D towards NADP+. Instead, when the residue aspartate at position 32 is mutated to a similar one, glutamate, this distance can be decreased to 2.10 Å (Fig. 2h) In this new mutant N32E/R33I/T34I, a new hydrogen bond is formed, increasing the binding with NAD+. Cyclic voltammetry results clearly show that both 6PGDHs produce significant oxidation current peaks at −0.3 V versus Ag/AgCl. The mutant N32E/R33I/T34I exhibits a current density 25% higher than that generated by the wild type (Fig. 4a). Too high temperature caused the deactivation of diaphorase, resulting in decreased power outputs in the biobattery

Discussion

Author Contributions

Additional Information