The apparent kinetic parameters were calculated by nonlinear regression using the program prism 5.0 (Prism, GraphPad Software, San Diego, CA). All kinetic parameters were obtained from at least three measurements. Effects of different metal ions (2 mM MnCl2, 2 mM MgCl2, 2 mM CaCl2, 2 mM CoCl2, 2 mM CuCl2, 2 mM ZnSO4, 2 mM NiSO4, 2 mM NaCl and 2 mM KCl) on the recombinant ZmIDH activity were also determined
using the standard assay method. X-ray structures of E. coli NADP-IDH (EcIDH, 9ICD), Bacillus subtilis NADP-IDH (BsIDH, 1HQS) and A. thiooxidans NAD-IDH (AtIDH, 2D4V) were downloaded www.selleckchem.com/products/Gefitinib.html from the pdb database (http://www.rcsb.org/pdb/). The ZmIDH model was generated using the swiss-model modeling server (http://swissmodel.expasy.org). Structure-based amino acid sequence alignment was conducted with clustalx program (ftp://ftp.ebi.ac.uk/pub/software/clustalw2) and espript 2.2 web tool (http://espript.ibcp.fr/ESPript/ ESPript/) (Gouet et al., 1999; Larkin et al., 2007). The cloned icd gene is 1263 bp in length, encoding a polypeptide of 420 amino acids. The overall GC content is about 46.4%, which is similar to that of the chromosomes of Zymomonas species (46–61%) (Seo et al., 2005). A homology search revealed that the deduced icd gene product shares 55%, 60% and 58% amino
acid identity with homodimeric IDHs from E. coli, B. subtilis NU7441 purchase and A. thiooxidans, respectively. The 3D-structure of ZmIDH was modeled using AtIDH (2D4V) as a template. A secondary structure-based alignment revealed that most structural elements were highly conserved 4-Aminobutyrate aminotransferase within prokaryotic homodimeric IDHs (Fig. 1). The amino acid residues involved in the binding of substrate and coenzyme were completely conserved (Fig. 1). The enzymatic interconversion of EcIDH between the catalytically active and inactive forms was regulated by IDH-kinase/phosphatase in response to changes in the metabolic environment (El-Mansi, 1998). Analogous sites corresponding to the phosphorylation site of EcIDH (Ser113)
were also found in AtIDH (Ser113), BsIDH (Ser104) and ZmIDH (Ser102) (Fig. 1), although there is no evidence that these three enzymes can be phosphorylated in vivo. The cofactor specificity of EcIDH was partially conferred by interactions between NADP+ and Lys344, Tyr345 and Val351 (Zhu et al., 2005). These residues were conserved in the NADP+-dependent BsIDH, but were replaced by Asp357, Ile358 and Ala364 in the NAD+-dependent AtIDH (Fig. 1). Asp357 was identified as the direct cofactor-specificity determinant, which discriminated NAD+ from NADP+ by forming double hydrogen bonds with the 2′- and 3′-hydroxyl groups of the adenosine ribose (Imada et al., 2008). The same amino acid residues were found in the corresponding sites of ZmIDH (Asp348, Ile349 and Ala355) (Fig. 1).