Display Settings:

Format

Send to:

Choose Destination
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
J Biol Chem. 2002 May 3;277(18):15613-20. Epub 2002 Feb 25.

Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity.

Author information

  • 1Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.

Abstract

Exchanging the identity of amino acids at four key locations within the Arabidopsis thaliana oleate desaturase (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) Science 282, 1315-1317). We report that four analogous substitutions 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 products 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 approximately 49-fold, and that residue 148 is also a predominant determinant of reaction outcome. Comparison of residues at position 148 of FAD2, LFAH, and the Ricinus oleate hydroxylase prompted us to rationally engineer LFAH-N149I, a variant with approximately 1.9-fold increase in hydroxylation specificity compared with that of wild-type LFAH. Control experiments showed that the wild-type Arabidopsis thaliana FAD2 desaturase has inherent, low level, hydroxylation activity. Further, fatty acid desaturases from different kingdoms and with different regiospecificities exhibit similar intrinsic hydroxylase activity, underscoring fundamental mechanistic similarities between desaturation and hydroxylation. For LFAH mutants the hydroxylation:desaturation ratio is 5-9-fold higher for 18-carbon versus 16-carbon substrates, supporting our hypothesis that substrate positioning in the active site plays a key role in the partitioning of catalytic specificity.

PMID:
11864983
[PubMed - indexed for MEDLINE]
Free full text

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire
    Loading ...
    Write to the Help Desk