Citation: HOU Shuhua, JI Debin, LIU Wujun, WANG Lei. Effect of Site-Directed Mutagenesis in Coenzyme-Binding Domain of Malic Enzyme on Coenzyme Activities for Nicotinamide Adenine Dinucleotide Analogs[J]. Chinese Journal of Applied Chemistry, ;2016, 33(4): 436-441. doi: 10.11944/j.issn.1000-0518.2016.04.150268 shu

Effect of Site-Directed Mutagenesis in Coenzyme-Binding Domain of Malic Enzyme on Coenzyme Activities for Nicotinamide Adenine Dinucleotide Analogs

HOU Shuhua ^a,, ,
JI Debin ^b ,
LIU Wujun ^b ,
WANG Lei ^b

1.
a Department of Chemistry, Bohai University, Jinzhou, Liaoning 121013, China;
2.
b Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, China

Corresponding author: HOU Shuhua,
Received Date: 28 July 2015
Available Online: 8 December 2015

The cofactor-binding domains of malic enzyme(ME) L310, Q401 and L404 were found to have a steric effect on binding of nicotinamide adenine dinucleotide(NAD⁺). The site-directed mutagenesis of these three sites shows that the cofactor activities of NAD⁺ analogs(B1~B7) change in some level, indicating that these sites have a critical role in binding the cofactors. A high-throughput screen between malic enzyme mutants and NAD⁺ analogs affords 2 different mutants capable of taking NAD⁺ analogs as the cofactor. ME-Q401H/L404T has a 50-fold higher k_cat/K_m than the that of wild-type ME for the analog B4. For ME-L310M/Q401N, the k_cat/K_m are 16-fold and 5-fold higher than those of wild-type ME for the cofactors B4 and B3. The coenzyme activity can be increased through site-directed mutagenesis of cofactor-binding domains.
- malic enzyme,
- site-directed mutagenesis,
- coenzyme activity
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