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

doi:10.11944/j.issn.1000-0518.2016.04.150268

Chinese Journal of Applied Chemistry ›› 2016, Vol. 33 ›› Issue (4): 436-441.DOI: 10.11944/j.issn.1000-0518.2016.04.150268

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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

^aDepartment of Chemistry,Bohai University,Jinzhou,Liaoning 121013,China
^bDalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian,Liaoning 116023,China

Received:2015-07-28 Accepted:2015-12-08 Published:2016-04-01 Online:2016-04-01
Contact: HOU Shuhua

Abstract

Abstract:

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.

Key words: malic enzyme, site-directed mutagenesis, coenzyme activity

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.

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

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