A single amino acid substitution in the FAD-binding domain causes the inactivation of isomerase.

We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in by ectopically expressing a isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a gene with a single-nucleotide mutation converting GCA to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val was not responsible for the enzyme inactivation in the AlaVal mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked AlaVal mutant. It is, therefore, reasonable to assume that Ala residue is critical for PAI functionality. pai: isomerase; CLA: conjugated linoleic acid; t10c12-CLA: trans 10, cis 12-CLA; LA: linoleic acid (18:2n-6); FAD: flavin adenine dinucleotide.