MUTATIONAL ANALYSIS OF A FATTY ACYL-COENZYME-A SYNTHETASE SIGNATURE MOTIF IDENTIFIES 7 AMINO-ACID-RESIDUES THAT MODULATE FATTY-ACID SUBSTRATE-SPECIFICITY

Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2. 1.3) catalyzes the formation of fatty acyl-CoA by a two-step process t hat proceeds through the hydrolysis of pyrophosphate. In Escherichia c oli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12-C18) and in the regulation of the global transcriptional r egulator FadR, The E. coli fatty acyl-CoA synthetase has remarkable am ino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancest ry, Most notable in this regard is a 26-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKEE, common to all fatty acyl-CoA synthetases for which sequence information is available, Within this consensus ar e 8 invariant and 13 highly conserved amino acid residues in the 12 fa tty acyl-CoA synthetases compared, We propose that this sequence repre sents the fatty acyl-CoA synthetase signature motif (FAGS signature mo tif). This region of fatty acyl-CoA synthetase from E. coli, (431)NGWL HTGDIAVMDEEGFL-RIVDRKK(455), contains 17 amino acid residues that are either identical or highly conserved to the FAGS signature motif. Eigh teen site directed mutations within the fatty acyl-CoA synthetase stru ctural gene (fadD) corresponding to this motif were constructed to eva luate the contribution of this region of the enzyme to catalytic activ ity, Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using c ell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild -type fatty acyl-CoA synthetase activity profiles; 2) those that had l ittle or no enzyme activity; and 3) those that resulted in lowering an d altering fatty acid chain length specificity, Among the 18 mutants c haracterized, 7 fall in the third class, We propose that the FAGS sign ature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose pa rt of the fatty acid binding site within the enzyme.