Poster: Evolution of developmental & physiological mechanisms

Abs # 820: Switching enzyme specificity by alternative subcellular targeting

Presenter:	Heilmann, Ingo , heilmann@bnl.gov
Authors	Heilmann, Ingo  (A)   Shanklin, John  (A)
Affiliations:	(A): Brookhaven National Laboratory

The unique functionality, substrate specificity, and regiospecificity of an enzyme typically evolves by gradual accumulation of changes in its catalytic portion until new properties arise. However, an emerging body of evidence suggests an enzyme’s functional characteristics can also depend on metabolic context, and temporal and spatial dynamics of enzyme interactions can determine enzyme function. The Arabidopsis desaturase (ADS) family comprises nine members, including the cytoplasmic Δ⁹-acyl-desaturases ADS1 and ADS2, and the Δ⁷-acyl-desaturase FAD5, which contains a plastidial transit peptide. When the cytoplasmic ADS1 and ADS2 were each retargeted to the plastid, and FAD5 to the cytoplasm, the regiospecificity for 16:0 desaturation in Arabidopsis seeds shifted dramatically in all three cases. Our data indicate overall regiospecificity shifts of 25-70-fold, with Δ⁷:Δ⁹ product ratios of ~1:13 when the desaturases were expressed without a transit peptide, and of ~2-5:1 when they were expressed with a transit peptide. Evidently, the Δ⁷- or Δ⁹-regiospecificities of ADS enzymes are determined by alternate subcellular targeting rather than by differences in primary sequence accumulated over the course of evolution. Coexpression of cucumber monogalactosyldiacylglycerol (MGDG) synthase with individual ADS enzymes (lacking transit peptides) in yeast resulted in Δ⁷-desaturation not normally seen in yeast. 16:1Δ⁷ was highly enriched in MGDG isolated from the coexpressors, while absent from yeast cultures expressing either MGDG synthase or an ADS enzyme alone. Thus, alternative enzyme targeting may provide access to location-specific substrate pools that determine novel specificities. Funded by DOE, OEA/DOW (JS) and DFG/Emmy-Noether (IH).