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.

[1]  P. Black Characterization of FadL-specific fatty acid binding in Escherichia coli. , 1990, Biochimica et biophysica acta.

[2]  B. Witholt,et al.  DNA sequence determination and functional characterization of the OCT‐plasmid‐encoded alkJKL genes of Pseudomonas oleovorans , 1992, Molecular microbiology.

[3]  A. Aderem,et al.  The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. , 1995, Trends in biochemical sciences.

[4]  L. Suzuki,et al.  Further purification, characterization and salt activation of acyl-CoA synthetase from Escherichia coli. , 1985, Biochimica et biophysica acta.

[5]  G. Gerber,et al.  Fatty acid uptake in Escherichia coli: regulation by recruitment of fatty acyl-CoA synthetase to the plasma membrane. , 1993, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[6]  P. Black,et al.  Bacterial long-chain fatty acid transport. Identification of amino acid residues within the outer membrane protein FadL required for activity. , 1993, The Journal of biological chemistry.

[7]  J. Kondo,et al.  Structure and regulation of rat long-chain acyl-CoA synthetase. , 1990, The Journal of biological chemistry.

[8]  P. Black,et al.  Transport of long-chain fatty acids in Escherichia coli. Evidence for role of fadL gene product as long-chain fatty acid receptor. , 1986, The Journal of biological chemistry.

[9]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[10]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[11]  H. Lodish,et al.  Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein , 1994, Cell.

[12]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[13]  B. Corkey,et al.  Long chain acyl coenzyme A and signaling in neutrophils. An inhibitor of acyl coenzyme A synthetase, triacsin C, inhibits superoxide anion generation and degranulation by human neutrophils. , 1994, The Journal of biological chemistry.

[14]  J. Gordon,et al.  Biochemical studies of three Saccharomyces cerevisiae acyl-CoA synthetases, Faa1p, Faa2p, and Faa3p. , 1994, The Journal of biological chemistry.

[15]  P. Black,et al.  Linker mutagenesis of a bacterial fatty acid transport protein. Identification of domains with functional importance. , 1991, The Journal of biological chemistry.

[16]  Stephen G. Tell,et al.  BioSCAN: a network sharable computational resource for searching biosequence databases , 1996, Comput. Appl. Biosci..

[17]  P. Black,et al.  Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. , 1994, Biochimica et biophysica acta.

[18]  P. Black,et al.  Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. , 1992, The Journal of biological chemistry.

[19]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[20]  J. Gordon,et al.  Genetic analysis of the role of Saccharomyces cerevisiae acyl-CoA synthetase genes in regulating protein N-myristoylation. , 1994, The Journal of biological chemistry.

[21]  J. Rothman,et al.  Possible role for fatty acyl-coenzyme A in intracellular protein transport , 1987, Nature.

[22]  J. Gordon,et al.  Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N- myristoylation , 1992, The Journal of cell biology.

[23]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[24]  P. Black Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport , 1991, Journal of bacteriology.

[25]  R. Simons,et al.  Transport of long-chain fatty acids by Escherichia coli: mapping and characterization of mutants in the fadL gene. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[26]  P. Groot,et al.  Fatty acid activation: specificity, localization, and function. , 1976, Advances in lipid research.

[27]  W. D. Nunn,et al.  Purification and characterization of acyl coenzyme A synthetase from Escherichia coli. , 1981, The Journal of biological chemistry.

[28]  P. Black,et al.  Evidence that His110 of the protein FadL in the outer membrane of Escherichia coli is involved in the binding and uptake of long-chain fatty acids: possible role of this residue in carboxylate binding. , 1995, The Biochemical journal.

[29]  J. Rothman,et al.  Fatty acyl-coenzyme a is required for budding of transport vesicles from Golgi cisternae , 1989, Cell.

[30]  C. DiRusso,et al.  Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A. , 1992, The Journal of biological chemistry.

[31]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[32]  E V Koonin,et al.  A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif. , 1993, Journal of molecular biology.

[33]  G. Gokel,et al.  Protein N-myristoylation. , 1991, The Journal of biological chemistry.

[34]  R. Dieckmann,et al.  Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases , 1995, FEBS letters.

[35]  P. Bentley,et al.  Potentiation of diacylglycerol-activated protein kinase C by acyl-coenzyme A thioesters of hypolipidaemic drugs. , 1989, Biochemical and biophysical research communications.

[36]  P. Babbitt,et al.  Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases. , 1992, Biochemistry.

[37]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[38]  N. Shimizu,et al.  Human long-chain acyl-CoA synthetase: structure and chromosomal location. , 1992, Journal of biochemistry.

[39]  P. Black,et al.  Use of transposon TnphoA to identify genes for cell envelope proteins of Escherichia coli required for long-chain fatty acid transport: the periplasmic protein Tsp potentiates long-chain fatty acid transport , 1994, Journal of bacteriology.

[40]  J. Walker,et al.  Distantly related sequences in the alpha‐ and beta‐subunits of ATP synthase, myosin, kinases and other ATP‐requiring enzymes and a common nucleotide binding fold. , 1982, The EMBO journal.

[41]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[42]  P. Black,et al.  Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli. , 1987, The Journal of biological chemistry.

[43]  D. E. Levin,et al.  Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N- myristoylation and cellular lipid metabolism , 1994, The Journal of cell biology.

[44]  Tokuo T. Yamamoto,et al.  Cloning and functional expression of a novel long-chain acyl-CoA synthetase expressed in brain. , 1992, Journal of biochemistry.