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Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

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Abstract

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K m values. Xt-F143Y caused a higher increase in the K m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.

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Authors and Affiliations

  1. United Graduate School of Agricultural Sciences, Tottori University, Koyama, Tottori, 680-8553, Japan

    Mohamed M. Eltayeb

  2. Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Koyama, Tottori, 680-8553, Japan

    Isam A. Mohamed Ahmed, Jiro Arima & Nobuhiro Mori

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  1. Mohamed M. Eltayeb
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  2. Isam A. Mohamed Ahmed
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  3. Jiro Arima
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  4. Nobuhiro Mori
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Correspondence to Jiro Arima.

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Eltayeb, M.M., Mohamed Ahmed, I.A., Arima, J. et al. Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase. Mol Biotechnol 55, 268–276 (2013). https://doi.org/10.1007/s12033-013-9678-0

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