期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 286, 期 15, 页码 12860-12869出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.191098
关键词
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资金
- CSIRO Office of the Chief Executive Post-doctoral Fellowship Scheme
Membrane-bound fatty acid desaturases and related enzymes play a pivotal role in the biosynthesis of unsaturated and various unusual fatty acids. Structural insights into the remarkable catalytic diversity and wide range of substrate specificities of this class of enzymes remain limited due to the lack of a crystal structure. To investigate the structural basis of the double bond positioning (regioselectivity) of the desaturation reaction in more detail, we relied on a combination of directed evolution in vitro and a powerful yeast complementation assay to screen for Delta x regioselectivity. After two selection rounds, variants of the bifunctional Delta 12/Delta 9-desaturase from the house cricket (Acheta domesticus) exhibited increased Delta 9-desaturation activity on shorter chain fatty acids. This change in specificity was the result of as few as three mutations, some of them near the putative active site. Subsequent analysis of individual substitutions revealed an important role of residue Phe-52 in facilitating Delta 9-desaturation of shorter chain acyl substrates and allowed for the redesign of the cricket Delta 12/Delta 9-desaturase into a 16:0-specific Delta 9-desaturase. Our results demonstrate that a minimal number of mutations can have a profound impact on the regiselectivity of acyl-CoA fatty acid desaturases and include the first biochemical data supporting the acyl-CoA acyl carrier specificity of a desaturase able to carry out Delta 12-desaturation.
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