University of Khartoum

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

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

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Title: Identification of Residues Essential for the Activity and Substrate Affinity of l- Carnitine Dehydrogenase
Author: Eltayeb, Mohamed M.; Mohamed Ahmed, Isam A.; Arima,Jiro; Mori,Nobuhiro
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 Km values. Xt-F143Y caused a higher increase in theKm 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 B 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDHand Phe143 of Xt-CDHare essential for substrate recognition.
URI: http://khartoumspace.uofk.edu/handle/123456789/21024
Date: 2016-05-04


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