Desaturation and Hydroxylation

Abstract

Exchanging the identity of amino acids at four key locations within the Arabidopsis thaliana oleate desatu- rase (FAD2) and the Lesquerella fendleri hydroxylase/ desaturase (LFAH) was shown to influence partitioning between desaturation and hydroxylation (Broun, P., Shanklin, J., Whittle, E., and Somerville, C. (1998) Sci- ence 282, 1315–1317). We report that four analogous sub- stitutions in the FAD2 sequence by their equivalents from the castor oleate hydroxylase result in hydroxy fatty acid accumulation in A. thaliana to the same levels as for the wild-type castor hydroxylase. We also describe the relative contribution of these substitutions, both individually and in combination, by analyzing the prod- ucts resulting from their expression in A. thaliana and/or Saccharomyces cerevisiae. Yeast expression showed that M324V, a change reachable by a single point mutation, altered the product distribution ⬃49- fold, and that residue 148 is also a predominant deter- minant of reaction outcome. Comparison of residues at position 148 of FAD2, LFAH, and the Ricinus oleate hy- droxylase prompted us to rationally engineer LFAH- N149I, a variant with ⬃1.9-fold increase in hydroxyla- tion specificity compared with that of wild-type LFAH. Control experiments showed that the wild-type Arabi- dopsis thaliana FAD2 desaturase has inherent, low level, hydroxylation activity. Further, fatty acid desatu- rases from different kingdoms and with different regio- specificities exhibit similar intrinsic hydroxylase activ- ity, underscoring fundamental mechanistic similarities between desaturation and hydroxylation. For LFAH mu- tants the hydroxylation:desaturation ratio is 5–9-fold higher for 18-carbon versus 16-carbon substrates, sup- porting our hypothesis that substrate positioning in the active site plays a key role in the partitioning of cata- lytic specificity. Hydroxy