Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase
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Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase Huri Bulut 1,2 & Busra Yuksel 3 & Mehmet Gul 3 & Meryem Eren 3 & Ersin Karatas 4 & Nazli Kara 1 & Berin Yilmazer 4 & Abdurrahim Kocyigit 2 & Nikolaos E. Labrou 5 & Baris Binay 6 Received: 25 May 2020 / Accepted: 18 September 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme’s thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies. Keywords Candida boidinii . Formate dehydrogenase . Site-directed mutagenesis . Thermostability . Molecular modeling
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003429-0) contains supplementary material, which is available to authorized users.
* Baris Binay [email protected] Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
Introduction The NAD+-dependent format dehydrogenase (EC 1.2.1.2; FDH) catalyzes the reversible conversion of formate (HCO2−) to carbon dioxide (CO2) using as coenzyme the NAD+/NADH system [1]. FDH was discovered in the 1950s and nowadays attracts the attention of sustainable biocatalysis and biotechnology [2]. It has become the focus of intense research since the formate formed from CO2 can be used as building block for the synthesis of various chemicals and as a store energy in fuel cells in the energy industry [3–7]. NAD+-dependent FDH genes have been identified in many different organisms and those enzymes have very close kinetic parameters. The genus Candida has attracted the most interest among these organisms because of the stability and better catalytic activity of the FDH enzyme they contain [8]. NAD+-depende
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