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Engineering d ‐Lactate Dehydrogenase to Favor an Non‐natural Cofactor Nicotinamide Cytosine Dinucleotide
Author(s) -
Liu Yuxue,
Li Qing,
Wang Lei,
Guo Xiaojia,
Wang Junting,
Wang Qian,
Zhao Zongbao K.
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900766
Subject(s) - nicotinamide adenine dinucleotide , nad+ kinase , cofactor , biochemistry , chemistry , lactate dehydrogenase , escherichia coli , protein engineering , enzyme , redox , mutagenesis , mutant , organic chemistry , gene
Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus ‐derived NAD‐dependent d ‐lactate dehydrogenase ( Lh DLDH) to favor NCD by semirational 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, thus suggesting the potential to construct NCD‐mediated orthogonal metabolism.