Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine

Rusnak, Frank; Sakaitani, Masahiro; Drueckhammer, Dale G.; Reichert, Janice; Walsh, Christopher T.

doi:10.1021/bi00225a027

Cited by 150 publications

(130 citation statements)

References 69 publications

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“…ORF 8 encodes a protein highly similar (Table 3) to DhbF from B. subtilis (Rowland et al, 1996), which is highly related to the E. coli EntF protein that participates in the final stages of enterobactin biosynthesis (Rusnak et al, 1991). RT-PCR experiments proved that this gene is part of a bicistronic message (Fig.…”

Section: Nucleotide Analysis Of Siderophore Biosynthetic and Utilizatmentioning

confidence: 94%

Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport

et al. 2003

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The Acinetobacter baumannii 8399 clinical isolate secretes dihydroxybenzoic acid (DHBA) and a high-affinity catechol siderophore, which is different from other bacterial iron chelators already characterized. Complementation assays with enterobactin-deficient Escherichia coli strains led to the isolation of a cosmid clone containing A. baumannii 8399 genes required for the biosynthesis and activation of DHBA. Accordingly, the cloned fragment harbours a dhbACEB polycistronic operon encoding predicted proteins highly similar to several bacterial proteins required for DHBA biosynthesis from chorismic acid. Genes encoding deduced proteins related to the E. coli Fes and the Bacillus subtilis DhbF proteins, and a putative Yersinia pestis phosphopantetheinyl transferase, all of them involved in the assembly and utilization of catechol siderophores in other bacteria, were found next to the dhbACEB locus. This A. baumannii 8399 gene cluster also contained the om73, p45 and p114 predicted genes encoding proteins potentially involved in transport of ferric siderophore complexes. The deduced products of the p114 and p45 genes are putative membrane proteins that belong to the RND and MFS efflux pump proteins, respectively. Interestingly, P45 is highly related to the E. coli P43 (EntS) protein that participates in the secretion of enterobactin. Although P114 is similar to other bacterial efflux pump proteins involved in antibiotic resistance, its genetic arrangement within this A. baumannii 8399 locus is different from that described in other bacteria. The product of om73 is a Fur-and iron-regulated surface-exposed outer-membrane protein. These characteristics together with the presence of a predicted TonB box and its high similarity to other siderophore receptors indicate that OM73 plays such a role in A. baumannii 8399. The 184 nt om73-p114 intergenic region contains promoter elements that could drive the expression of these divergently transcribed genes, all of which are in close proximity to almost perfect Fur boxes. This arrangement explains the iron-and Fur-regulated expression of om73, and provides strong evidence for a similar regulation for the expression of p114. INTRODUCTIONBacteria survive and multiply under iron-limiting conditions, such as those found in natural and medical environments, by expressing active systems that gather this micronutrient, which is essential for most micro-organisms with some exceptions such as lactobacilli. Some systems involve the secretion of low-molecular-mass ferric-binding compounds called siderophores, which can be classified into different categories based on their chemical structure (Neilands, 1981(Neilands, , 1995. Many of these high-affinity ironchelating molecules contain catecholate groups that are part of the iron-binding site. Enterobactin, the prototype catechol siderophore produced by Escherichia coli, is a cyclic trimer of 2,3-dihydroxybenzoyl-L-serine (O'Brien & Gibson, 1970;Walsh et al., 1990). In contrast, siderophores such as vibriobactin and anguibactin, produced by...

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Section: Nucleotide Analysis Of Siderophore Biosynthetic and Utilizatmentioning

confidence: 94%

Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport

et al. 2003

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“…strain M114, and this is discussed below. Significant similarity was also found to a family of enzymes including GrsB (53), TycA (57), SrfA (10), AcvA, PcbA, and PcbB (8,27,40), EntF (37), and AngR (14,52). These enzymes are peptide synthetases, with the last two being involved in siderophore synthesis.…”

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confidence: 92%

“…The C-terminal segment of PvdD (336 amino acids), which is not part of either repeat sequence, was independently compared with sequences in the translated GenBank database. The only significant similarities were to EntF from E. coli (37) and the AcvA protein from Penicillium chrysogenum (40), both of which are peptide synthetases. In each case, similarity was centered around a thioesterase active-site motif (GXSXG [20], where X is any amino acid) which is present at residues 2240 to 2244 in PvdD.…”

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confidence: 99%

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

1995

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Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.Pseudomonas aeruginosa is an opportunistic pathogen which infects injured, immunodeficient, or otherwise compromised patients. The bacteria secrete a siderophore, pyoverdine, which is likely to play an important role in infection by competing with transferrin for iron in order to overcome the iron-withholding defense mechanism present in mammals (3,11,42). Almost all isolates of P. aeruginosa from infected patients secrete pyoverdine (9,22,29), and it has been shown that pyoverdine is present in the sputa of cystic fibrosis patients infected with P. aeruginosa (21). A mutant of P. aeruginosa which is unable to synthesize pyoverdine showed reduced virulence in an animal model of infection (24).Pyoverdine from P. aeruginosa PAO is a water-soluble, yellow-green fluorescent compound. It consists of a dihydroxyquinoline chromophoric group linked to an eight-residuevia the N-terminal serine, with a small dicarboxylic acid attached to the chromophore. Iron complexation is thought to occur through oxygen atoms present on the dihydroxyquinoline and two hydroxamate units supplied by the L-N 5 -OH-Orn residues. Several Pseudomonas species produce similar compounds, variously called pyoverdines or pseudobactins, all of which have the same dihydroxyquinoline group but differ in the nature of the attached peptide (reviewed in references 1 and 7), and it is likely that all of these are synthesized by similar mechanisms. Synthesis of the chromophoric group begins with condensation of D-tyrosine and L-2,4-diaminobutyric acid (7), with glutamic acid being the precursor of the amide-linked dicarboxylic acid (50). It has been suggested that biosynthesis of the peptide moiety of pyoverdine occurs by a nonribosomal mechanism (30).Little is known about the molecular nature of enzymes involved in the biosynthesis of pyoverdine in P. aeruginosa or any other pseudomonad. Recently, pvdA, which encodes an enzyme (L-ornithine N 5 -oxygenase) responsible for catalyzing the hydroxylation of ornithine, an early step in pyoverdine biosynthesis in P. aeruginosa, has been characterized at the molecular level (55). The pbsC gene, which is involved in synthesis of pseudobactin by Pseudomonas sp. strain M114, has also been seque...

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“…Our major focus was on the assembly of nonribosomal peptides. This started, on the one hand, with how the antibiotic vancomycin was put together (32) in the hope of eventually engineering a version that would work against VRE and, on the other hand, with the E. coli virulence factor, the siderophore enterobactin, to try to understand how three amide and three ester bonds could be built and put into a cyclic framework by one enzyme (EntF) (33).…”

Section: Enterobactin Synthase Is a Two-module Nonribosomal Peptide Smentioning

confidence: 99%

At the Intersection of Chemistry, Biology, and Medicine

Walsh

2017

Annu. Rev. Biochem.

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After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 10 5 to 10 6 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.

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Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine

Cited by 150 publications

References 69 publications

Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport

Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

At the Intersection of Chemistry, Biology, and Medicine

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