Engineering D-lactate dehydrogenase to favor a non-natural cofactor nicotinamide cytosine dinucleotide.

Synthetic nicotinamide adenine dinucleotide (NAD) analogues, are of great scientific and biotechnological interests. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied in creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. Herein, we engineered Lactobacillus helveticus derived NAD-dependent D-lactate dehydrogenase ( Lh DLDH) to favor NCD by semi-rational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a "gate" guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant Lh DLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD-preference afforded much less D-lactate, suggesting the potential to construct NCD-mediated orthogonal metabolism.