Further purification, characterization and salt activation of acyl-CoA synthetase fromEscherichia coli

Kameda, Kensuke; Suzuki, Lidia K.; Imai, Yoh

doi:10.1016/0304-4165(85)90158-8

Cited by 21 publications

(16 citation statements)

References 19 publications

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“…This enzyme shares amino acid sequence similarities to other fatty acyl-CoA synthetases and more broadly the family of adenylate-forming enzymes (26 -28). The placement of this enzyme in the family of adenylate-forming enzymes is based on amino acid similarities within a proposed AMP/ATP binding domain (25). Of particular relevance to the present work was the identification of a second conserved segment that appears to be restricted to the family of fatty acyl-CoA synthetases (26).…”

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confidence: 86%

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Mutational Analysis of a Fatty Acyl-Coenzyme A Synthetase Signature Motif Identifies Seven Amino Acid Residues That Modulate Fatty Acid Substrate Specificity

Black

Zhang²,

Weimar

et al. 1997

Journal of Biological Chemistry

148

157

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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, DGWLHTGDIGXWX-PXGXLKIIDRKK, 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, 431 NGWLHTGDIAVMDEEGFL-RIVDRKK 455 , 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.

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confidence: 86%

“…coli contains a single fatty acyl-CoA synthetase with broad chain length specificity for saturated, unsaturated, and polyunsaturated fatty acids (24,25). This enzyme is essential for the activation of exogenous long chain fatty acids destined for ␤-oxidation and plays an essential role in the regulation of the transcription factor FadR.…”

Section: Discussionmentioning

confidence: 99%

Mutational Analysis of a Fatty Acyl-Coenzyme A Synthetase Signature Motif Identifies Seven Amino Acid Residues That Modulate Fatty Acid Substrate Specificity

Black

Zhang²,

Weimar

et al. 1997

Journal of Biological Chemistry

148

157

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“…The salt activation of another acyl-CoA synthetase from E. coli was described previously (49). The reaction rate of this E. coli enzyme depends on the salt concentration, with 2.5-fold enhancement of the enzyme activity being observed on the addition of 100 mM (NH 4 ) 2 SO 4 .…”

Section: Discussionmentioning

confidence: 99%

Nitrile Pathway Involving Acyl-CoA Synthetase

Hashimoto

Hosaka

Oinuma

et al. 2005

Journal of Biological Chemistry

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Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acylCoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH 2 -terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent K m values for butyric acid, CoA, and ATP were 0.32 ؎ 0.04, 0.37 ؎ 0.02, and 0.22 ؎ 0.02 mM, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (k cat /K m ) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime3nitrile3amide3acid3acyl-CoA) comprising these enzymes.

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“…This observation probably accounts for the discrepancies in the size predicted by the nucleic acid sequence [7,8] and that of the purified protein from wildtype E. coli [6,25]. Proteolysis appears to be mediated by the outer membrane protease OmpT, and does not play a role in the processing of FadD in i o.…”

Section: Discussionmentioning

confidence: 99%

Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT

YOO

CHENG

Gerber

2001

Biochemical Journal

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Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase [also known as fatty acid :CoA ligase (AMPforming) ; EC 6.2.1.3], as a 42-50 kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62 kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62 kDa FadD enzyme is a substrate for the outer membrane protease OmpT in itro, producing a 43 kDa Cterminal fragment and a 19 kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62 kDa form of the enzyme is present in i o, but we utilized the proteolytic sensitivity of FadD to investigate its structure. Photoaffinity labelling experiments revealed that both intact FadD and the 43 kDa fragment bound a long-chain fatty acid.

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Further purification, characterization and salt activation of acyl-CoA synthetase fromEscherichia coli

Cited by 21 publications

References 19 publications

Mutational Analysis of a Fatty Acyl-Coenzyme A Synthetase Signature Motif Identifies Seven Amino Acid Residues That Modulate Fatty Acid Substrate Specificity

Mutational Analysis of a Fatty Acyl-Coenzyme A Synthetase Signature Motif Identifies Seven Amino Acid Residues That Modulate Fatty Acid Substrate Specificity

Nitrile Pathway Involving Acyl-CoA Synthetase

Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT

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