Abstract
On the basis of a phylogenic tree constructed using the available Arabidopsis thaliana genomic sequence, new fatty acid (FA) omega-hydroxylases were cloned and expressed in yeast. Several uncharacterized cytochrome P450s (CYP, P450), comprising a phylogenic sub-cluster (CYP86A, 94B, 94C, 96A, 704A, 97B and 711A) demonstrated FA omega-hydroxylase activities towards saturated FAs with medium chain length. While CYP96A4 showed the highest catalytic activity among the enzymes characterized in this study, other CYP96A subfamily members did not display any potent activity. In addition, CYP704A2 and CYP711A1, which are phylogenetically distant to both CYP86 and CYP94 with FA omega-hydroxylase activities, did not show any metabolic conversion of FAs. As we have studied omega-hydroxylation of FAs catalyzed by a series of CYP94 isoforms, active site models were produced to compare inactive CYP704A2 and CYP711A1 to active CYP94C1. The modeled structures revealed that the hydrophobicity in the heme binding site is very different between active and inactive isoforms. CYP94C1 contains highly hydrophobic residues while CYP704A2 and CYP711A1 do not. Our study provides evidence that substrate specificity is conserved in phylogenetically related isoforms and suggests specific residues in the active site pocket that play a key role in determining substrate specificity.
Published Version
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