scholarly journals Expression Cloning of a Pseudomonas Gene Encoding a Hydroxydecanoyl-Acyl Carrier Protein-Dependent UDP-GlcNAc Acyltransferase

1998 ◽  
Vol 180 (2) ◽  
pp. 330-337 ◽  
Author(s):  
Garry D. Dotson ◽  
Igor A. Kaltashov ◽  
Robert J. Cotter ◽  
Christian R. H. Raetz
Keyword(s):  
Lipid A ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Expression Cloning ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Lipid A Biosynthesis

UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of thelpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed inEscherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert ofP. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predictedPseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complementedE. coli RO138, a temperature-sensitive mutant harboringlpxA2. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]−1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′.

scholarly journals Structure of a Specialized Acyl Carrier Protein Essential for Lipid A Biosynthesis with Very Long-Chain Fatty Acids in Open and Closed Conformations

Biochemistry ◽  
2012 ◽  
Vol 51 (37) ◽  
pp. 7239-7249 ◽  
Author(s):  
Theresa A. Ramelot ◽  
Paolo Rossi ◽  
Farhad Forouhar ◽  
Hsiau-Wei Lee ◽  
Yunhuang Yang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid A ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Long Chain Fatty Acids ◽  
Lipid A Biosynthesis ◽  
Long Chain ◽  
Chain Fatty Acids

scholarly journals The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

2003 ◽  
Vol 185 (5) ◽  
pp. 1582-1589 ◽  
Author(s):  
Sean W. Jordan ◽  
John E. Cronan,
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Octanoic Acid ◽  
Acyl Carrier Protein ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Genomic Databases ◽  
E Coli ◽  
Lipoyl Domain

ABSTRACT In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.

scholarly journals Vibrio cholerae FabV Defines a New Class of Enoyl-Acyl Carrier Protein Reductase

2007 ◽  
Vol 283 (3) ◽  
pp. 1308-1316 ◽  
Author(s):  
R. Prisca Massengo-Tiassé ◽  
John E. Cronan
Keyword(s):  
Fatty Acid ◽  
Vibrio Cholerae ◽  
Mutant Strain ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Gene Encoding ◽  
E Coli ◽  
New Class ◽  
Enoyl Acp Reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.

scholarly journals Mutants of Escherichia coli with Temperature-sensitive Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

1974 ◽  
Vol 249 (23) ◽  
pp. 7468-7475
Author(s):  
Mark E. Harder ◽  
Ruth C. Ladenson ◽  
Steven D. Schimmel ◽  
David F. Silbert
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Acyl Carrier Protein ◽  
Temperature Sensitive ◽  
Carrier Protein

scholarly journals Structure of a SLC26 Anion Transporter STAS Domain in Complex with Acyl Carrier Protein: Implications for E. coli YchM in Fatty Acid Metabolism

Structure ◽  
2010 ◽  
Vol 18 (11) ◽  
pp. 1450-1462 ◽  
Author(s):  
Mohan Babu ◽  
Jack F. Greenblatt ◽  
Andrew Emili ◽  
Natalie C.J. Strynadka ◽  
Reinhart A.F. Reithmeier ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
E Coli ◽  
Anion Transporter ◽  
Stas Domain

scholarly journals Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

1993 ◽  
Vol 294 (2) ◽  
pp. 521-527 ◽  
Author(s):  
S A Morris ◽  
W P Revill ◽  
J Staunton ◽  
P F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Acyl Carrier Protein ◽  
Saccharopolyspora Erythraea ◽  
Carrier Protein ◽  
Freezing And Thawing ◽  
Acyl Carrier Proteins ◽  
Expression Plasmid ◽  
E Coli ◽  
Protein Dimer

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

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