Alanine-scanning mutation approach for classification of the roles of conserved residues in the activity and substrate affinity of L-carnitine dehydrogenase

作者：Eltayeb Mohamed M; Arima Jiro^*; Mori Nobuhiro

来源：Biotechnology Letters, 2014, 36(2): 309-317.

DOI：10.1007/s10529-013-1357-5

• Alanine-scanning mutation
• L-Carnitine dehydrogenase
• L-Carnitine estimation
• Substrate affinity
• Xanthomonas translucens

l-Carnitine dehydrogenase (CDH) is as an excellent tool for l-carnitine (l-Car) estimation. To date, four CDHs have been identified, that share 45 % homology of their proteins. Here 42 conserved residues of CDH from Xanthomonas translucens (Xt-CDH) were substituted successively with alanine. The resultant mutants were analyzed for catalytic activity. Active mutants were evaluated for their influence on l-Car affinity. Twenty-three mutants with reduced affinity toward l-Car were subjected to detailed kinetic analysis. Analytical data implied that all mutants had increased K (m) values. The mutants of R193A, E196A, W199A, R200A, F249A, and F253A that produced the greatest l-Car affinity disruption (K (m) %26gt; 200-folds of Xt-CDH) clustered near the putative active site. This information can provide a solid basis for the rational design of mutagenic investigation to improve CDHs.

• 出版日期2014-2