Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site.

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP-forming, EC ) catalyzes the esterification of fatty acids to CoA thioesters for further metabolism and is hypothesized to play a pivotal role in the coupled transport and activation of exogenous long-chain fatty acids in Escherichia coli. Previous work on the bacterial enzyme identified a highly conserved region (FACS signature motif) common to long- and medium-chain acyl-CoA synthetases, which appears to contribute to the fatty acid binding pocket. In an effort to further define the fatty acid-binding domain within this enzyme, we employed the affinity labeled long-chain fatty acid [(3)H]9-p-azidophenoxy nonanoic acid (APNA) to specifically modify the E. coli FACS. [(3)H]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing concentrations of palmitate. The site of APNA modification was identified by digestion of [(3)H]APNA cross-linked FACS with trypsin and separation and purification of the resultant peptides using reverse phase high performance liquid chromatography. One specific (3)H-labeled peptide, T33, was identified and following purification subjected to NH(2)-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK, which corresponded to residues 422 to 430 of FACS. This peptide is immediately adjacent to the region of the enzyme that contains the FACS signature motif (residues 431-455). This work represents the first direct identification of the carboxyl-containing substrate-binding domain within the adenylate-forming family of enzymes. The structural model for the E. coli FACS predicts this motif lies within a cleft separating two distinct domains of the enzyme and is adjacent to a region that contains the AMP/ATP signature motif, which together are likely to represent the catalytic core of the enzyme.

Mutational analysis of a fatty acyl-coenzyme A synthetase signature motif identifies seven 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 that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12-C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWXPXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431NGWLHTGDIAVMDEEGFLRIVDRKK455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell 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 little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.