Improvement of the soy formate dehydrogenase properties  by rational design

Kargov, I.S.; Kleimenov, S.Y.; Savin, S.S.; Tishkov, V.I.; Alekseeva, A.A.

Авторы: Kargov I.S., Kleimenov S.Y., Savin S.S., Tishkov V.I., Alekseeva A.A.
Журнал: Protein Engineering, Design and Selection
Том: 28
Номер: 6
Год издания: 2015
Издательство: Oxford University Press
Местоположение издательства: United Kingdom
Первая страница: 171
Последняя страница: 178
DOI: 10.1093/protein/gzv007
Аннотация: Previous experiments on substitution of the residue Phe290 to Asp, Asn and Ser in NAD+-dependent formate dehydrogenase from soya Glycine max (SoyFDH) showed important role of the residue in enzyme thermal stability and catalytic properties (Alekseeva et al., 2012a). In this work we continued site-directed mutagenesis experiments of the Phe290 and the residue was changed to Ala, Thr, Tyr, Glu, and Gln. All amino acid changes resulted in increase of catalytic constant from 2.9 to 3.5-4.7 s-1. The substitution Phe190Ala led to KMNAD+ decrease from 13.3 to 8.6 μM, and substitutions Phe190Tyr and Phe190Glu resulted in decrease and increase of KMHCOO- from 1.5 to 0.9 and-2.9 mM, respectively. The highest improvement of catalytic properties was observed for SoyFDH Phe190Ala which showed 2-fold higher catalytic efficiency with both substrates. Stability of mutants was examined by study of thermal inactivation kinetics and differential scanning calorimetry. All five amino acids provided increase of thermal stability of mutant SoyFDH in comparison with wild-type enzyme. Mutant SoyFDH Phe290Glu showed the highest improvement – the stabilization effect was 43 at 56оС. The DSC data agree with results of thermal inactivation kinetics. Substitutions Phe290Tyr, Phe290Thr, Phe290Gln, and Phe290Glu provided Tm value increase 0.6 - 6.6 degrees. SoyFDH Phe190Glu and previously prepared SoyFDH Phe190Asp show similar thermal stability as enzymes from Candida boidinii and Mycobacterium vaccae N10 and have higher catalytic efficiency with NAD+ compared to all described formate dehydrogenases. Therefore, these mutants are very perspective enzymes for coenzyme regeneration in processes of chiral synthesis with dehydrogenases.
Добавил в систему: Тишков Владимир Иванович

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Improvement of the soy formate dehydrogenase properties by rational designстатья

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