Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.

Stenflo, Johan; Holme, Elisabeth; Lindstedt, Sven; Chandramouli, Nagarajan; Huang, Linda H.; Tam, James P.; Merrifield, R. B.

doi:10.1073/pnas.86.2.444

Cited by 88 publications

(60 citation statements)

References 36 publications

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“…4). Other members of the Fe(II)/␣-KG-dependent enzyme family, among which are prolyl-4-hydroxylase and lysyl-5-hydroxylase, hydroxylate their substrates with a minimum chain length of six amino acids containing an Xaa-Pro/Lys-Gly sequence (44). Our results suggest that the recognition sequence for hydroxylation by DurX is similar (XaaAsp-Gly).…”

Section: Resultsmentioning

confidence: 58%

Insights into the Biosynthesis of Duramycin

Huo

Ökesli

Zhao

et al. 2017

Appl Environ Microbiol

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Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are characterized by the thioether cross-linked bisamino acids lanthionine (Lan) and methyllanthionine (MeLan). Duramycin contains 19 amino acids, including one Lan and two MeLans, an unusual lysinoalanine (Lal) bridge formed from the -amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-L-aspartic acid at position 15. These modifications are important for the interactions of duramycin with its biological target, phosphatidylethanolamine (PE). Based on the binding affinity and specificity for PE, duramycin has been investigated as a potential therapeutic, as a molecular probe to investigate the role and localization of PE in biological systems, and to block viral entry into mammalian cells. In this study, we identified the duramycin biosynthetic gene cluster by genome sequencing of Streptomyces cinnamoneus ATCC 12686 and investigated the dur biosynthetic machinery by heterologous expression in Escherichia coli. In addition, the analog duramycin C, containing six amino acid changes compared to duramycin, was successfully generated in E. coli. The substrate recognition motif of DurX, an ␣-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide DurA, was also investigated using mutagenesis of the DurA peptide. Both in vivo and in vitro results demonstrated that Gly16 is important for DurX activity.IMPORTANCE Duramycin is a natural product produced by certain bacteria that binds to phosphatidylethanolamine (PE). Because PE is involved in many cellular processes, duramycin is an antibiotic that kills bacteria, but it has also been used as a molecular probe to detect PE and monitor its localization in mammalian cells and even whole organisms, and it was recently shown to display broad-spectrum inhibition of viral entry into host cells. In addition, the molecule has been evaluated as treatment for cystic fibrosis. We report here the genes that are involved in duramycin biosynthesis, and we produced duramycin by expressing those genes in Escherichia coli. We show that duramycin analogs can also be produced. The ability to access duramycin and analogs by production in E. coli opens opportunities to improve duramycin as an antibiotic, PE probe, antiviral, or cystic fibrosis therapeutic.KEYWORDS antibiotic, antiviral agents, biosynthesis, duramycin, phosphatidylethanolamine L antibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) exhibiting antimicrobial activity. They are characterized by the thioether cross-linked amino acids lanthionine (Lan) and/or methyllanthionine (MeLan) (1), which are formed by dehydration of Ser and Thr residues, followed by intramolecular Michaeltype addition of Cys thiols to the resulting dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively (2-4) (Fig. 1a). These posttranslational modifications take place in the core peptide region of the precursor...

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Section: Resultsmentioning

confidence: 58%

Insights into the Biosynthesis of Duramycin

Huo

Ökesli

Zhao

et al. 2017

Appl Environ Microbiol

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“…Of the various dioxygenases used to search the microbial data bases, only the mammalian aspartyl/asparaginyl ␤-hydroxylase yielded the desired pattern. This enzyme hydroxylates certain aspartyl and asparaginyl residues in clotting factors and other proteins (60,61). Aspartyl/asparaginyl ␤-hydroxylase (33,34,60,61) belongs to the larger family of Fe 2ϩ /␣-ketoglutarate-dependent dioxygenases, which include prolyl and lysyl hydroxylases (62), deacetoxycephalosporin C synthase (35), taurine hydroxylase (56), and thymine hydroxylase (63,64).…”

Section: Discussionmentioning

confidence: 99%

Oxygen Requirement for the Biosynthesis of theS-2-Hydroxymyristate Moiety in Salmonella typhimurium Lipid A

Gibbons¹,

Lin²,

Cotter³

et al. 2000

Journal of Biological Chemistry

154

184

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Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O 2 . Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrixassisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of 18 O 2 confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/ asparaginyl ␤-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimurium aspartyl/asparaginyl ␤-hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O 2 -dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe 2؉ /␣-ketoglutaratedependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.Salmonella typhimurium and related organisms are enteric Gram-negative pathogens. In human hosts, S. typhimurium infection causes gastroenteritis, but in mice, the outcome is a fatal, typhoid-like sepsis, characterized by dissemination of bacteria into spleen, liver, and blood (1). S. typhimurium initially invade intestinal epithelial cells and M cells of Peyer's patches and then pass into the lymphatic system by colonizing phagocytic cells (1). The bacteria survive and multiply within modified vacuoles of macrophages (2) and gradually induce macrophage apoptosis (3-5).The ability of S. typhimurium to adapt to the acidic pH and the low divalent cation concentrations found inside macrophage vacuoles is critical to the infection process (6). The low Mg 2ϩ concentration within phagolysosomes activates the PhoP/PhoQ two-component signal transduction system of S. typhimurium, triggering numerous responses needed for survival and persistence within macrophages (7). Phosphorylation of the transcriptional regulator PhoP (8) by the sensory kinase PhoQ under such conditions results in the activation or...

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“…They are involved in a broad spectrum of primary and secondary biosynthetic pathways including the post-translational hydroxylation of proline and lysine residues in procollagens (Kivirikko et al, 1989), the biosynthesis of carnitine ) and blood-coagulation-factor VII (Stenflo et al, 1989) in mammals, the conversion of thymidine into uracil (Holme et al, 1970(Holme et al, , 1971Bankel et al, 1977) and of penicillins into cephalosporins (Baldwin and Abraham, 1988) in bacteria and fungi, the biosynthesis of clavulanic acid (Elson et al, 1987) and the macrolide antibioticum tylosin (Omura et al, 1984) in fungi, the formation of hydroxyproline-rich glycoproteins in plants (Chrispeels, 1969;Tanaka et al, 1980) and the biosynthesis of plant secondary products such a5 flavonoids Britsch et al, 1981), the alkaloids scopolamine (Hashimot0 and Yamada, 1986) and vindoline (De Carolis et al, 1990) and gibberellins (Hedden and Graebe, 1982).…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of flayanone 3β‐hydroxylases

Britsch

Dedio²,

Saedler³

et al. 1993

European Journal of Biochemistry

101

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A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3P-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callisteplzus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from l ? hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53 % identity) of flavanone 3P-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(11) enzymes demonstrated the strict genetic conservation of 14 anuno acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of tlavanone 3P-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.Flavanone 3P-hydroxylase (FHT) catalyzes 3P-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols [( +)-dihydroflavonols] which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins in plants (Heller and Forkmann, 1988;Forkmann, 1991). The enzyme from Petunia hybrida has been purified to homogeneity and characterized as a typical 2-oxoglutarate-dependent dioxygenase (Britsch and Grisebach, 1986;Britsch, 1990). Recently, we have isolated a near-full-length FHTencoding cDNA from a Petunia library (Britsch et al., 1992). Heterologous expression of the cDNA in Escherichia coli led to the formation of highly active recombinant FHT, thereby verifying the identity of the cDNA clone.Antibodies against FHT from Petunia readily crossreact with FHT from a number of other sources and were used to characterize specific FHT mutants (Britsch, 1990; Koch, 1992). Therefore, structural similarities of the FHT protein from different plants can be assumed. A comparison of the deduced amino acid sequence of the Petunia FHT with the sequence of FHT from barley (Meldgaard, 1992) indeed showed a high degree of identity (75%; Britsch et al., 1992).Consequently, the FHT cDNA from Petunia should serve as a useful heterologous probe to clone this enzyme from other plant sources. Utilizing the 945-bp EcoRI fragment from Petunia FHT as a hybridization probe, we were able to clone the corresponding cDNAs from carnation, china aster and stock. In this study, we report the cloning and sequence analysis of these particular FHT enzymes, the analysis of the consensus sequence derived from six individual FHT sequences and the modification of purified recombinant enzyme with diethyl pyrocarbonate, performed to elucidate the possible function of strictly conserved histidines. Furthermore, the sequences were also compared with other known 2-oxoglutarate-dependent dioxygenases f...

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Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.

Cited by 88 publications

References 36 publications

Insights into the Biosynthesis of Duramycin

Insights into the Biosynthesis of Duramycin

Oxygen Requirement for the Biosynthesis of theS-2-Hydroxymyristate Moiety in Salmonella typhimurium Lipid A

Molecular characterization of flayanone 3β‐hydroxylases

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