6‐Deoxyerythronolide‐B synthase 2 from <i>Saccharopolyspora erythraea</i>

Bevitt, Debra J.; Cortés, Jesús M.; Haydock, Stephen; Leadlay, Peter F.

doi:10.1111/j.1432-1033.1992.tb16603.x

Cited by 170 publications

(102 citation statements)

References 57 publications

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“…4 indicate that the region comprising motifs A-I is an N-terminal domain catalyzing the formation of acyl adenylates, which is followed by a cofactor binding domain concluded by certain similarities to acyl carrier proteins likewise binding 4'-phosphopantetheine. Surprisingly the proposed linker region between these domains did not show the Ala/Pro/Glu-enriched sequences detected in fatty acid synthases [24], polyketide synthases [25] or 2-oxodehydrogenase cofactor binding domains [26]. In an analysis of domain linker regions, a preference of certain amino acids has been confirmed [27], including Thr, Ser, Pro, Asp, Gly, while others are rare (Trp, Cys, His, Phe, Met).…”

Section: Discussionmentioning

confidence: 99%

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

et al. 1995

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Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32p]pp~ exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.Key words." Tyrocidine synthetase; Multienzyme; Peptide synthetase; Acyl-CoA-synthetase; Pantetheine; CoA structed of modules, each contributing one amino-or hydroxy acid to the peptide structure. Each module contains an adenylate-forming domain, a cofactor binding site, and a condensation/modification domain. The adenylate forming domains display significant similarity to acyl-CoA-synthetases and related proteins. We have predicted, by alignment of sequences of these three classes and those of acyl carrier proteins also binding 4'-phosphopantetheine, the boundaries of adenylate and cofactor domains. The linker region is not rich in Ala, Pro and Glu residues, as in fatty acid synthases and 2-oxo-acid dehydrogenases containing similar cofactor domains. Still, amino acids frequently found in linker regions are dominating. Upon deletion of the cofactor and following functional domains of tyrocidine synthetase 1, a stable N-terminal fragment was obtained which catalysed adenylate formation with similar kinetic properties as the apo-form of the peptide synthetase [8].

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Section: Discussionmentioning

confidence: 99%

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

et al. 1995

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“…Until now, the only evidence for the existence of three unusually large polypeptides as components of the erythromycin-producing polyketide synthase (DEBS) has been provided by DNA sequence analysis [6,7,8].…”

Section: Discussionmentioning

confidence: 99%

“…Further sequencing of eryA genes [7,8] has amply supported both the idea of 'an enyme for every move' (results quoted in [10]) and the likely domain organisaties of the synthase [6], with thr~ unusually large multienzyme polypeptides (DEB$1, DEBS 2 and DEBS 3) housing a total of at least 28 distinct active sites. Targetted deletion in rive of the ketoreductase domain in the N-terminal half of DEBS 3 [7] led to the accumulation of the expected metabolite, both confirming the identification of this domain and strengthening the prospects for rational modification of polyketide antibiotics.…”

Section: Introductionmentioning

confidence: 99%

“…The nature of the polyketide synthase that catalyses this multistep process, 6-deoxyerythronolide B synthase (DEBS), has been extensively investigated by genetic analysis [3][4][5] and by nucleotide sequencing of the structural genes encoding the synthase [6][7][8]. These eryA genes are found clustered near e~'mE, the erythromycin resistance gene [9], and extend over approximately 45 kilobase-pairs (kbp) of DNA.…”

Section: Introductionmentioning

confidence: 99%

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Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin‐producing polyketide synthase from Saccharopolyspora erythraea

Caffrey¹,

Bevitt²,

Staunton³

et al. 1992

FEBS Letters

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137

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The erA' A resion of the ¢rythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previo-sl~t been shown to contain thr~e large open reading frames (ORFs) that encode the components of 6-deoxyerythronolide B synthase (DEBS). Pol;,clonal antibodies were raised against recombinant proteins obtained by overexpression of 3' regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different hish molecular weight protein in extracts of erythromycin-producins S. erythraea cells. These putative DEB$ 2 and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis, The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences ofORFs 2 and 3. A third high molecular weight protein co.purified with DEBS 2 and DEBS 3 and had an N-terminal sequence that matched a protein sequence translated from the DNA sequen~ some 155 base pairs upstream from the previously proposed start codon of ORFI.

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“…The reason that FkbA ACP and enoyl reductase domains are more related to products of a heterologous gene rather than those belonging to the same gene is not clear at the present time. Apart from rapamycin, the most similar sequences to FkbA domains are those of the erythromycin PKS from Succhuropolysporu erythrueu [23,241, the mycoserosic acid synthase from Mycobacterium tuberculosis [26], the oleandomycin PKS from Streptomyces untibioticus [27], and the soraphen A PKS produced by Sorungium cellulosum [28].…”

Section: Discussionmentioning

confidence: 99%

Structural Organization of A Multifunctional Polyketide Synthase Involved in the Biosynthesis of the Macrolide Immunosuppressant FK506

Motamedi

Cai

Shafiee

et al. 1997

European Journal of Biochemistry

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The immunosuppressant FK506 is a 23-membered macrocyclic polyketide produced by several Streptomyces species. Sequencing of a 19.5-kb contiguous segment of DNA from the FK506 gene cluster of Streptomyces sp. MA6548 revealed the presence of a single 19.3-kb open reading frame designatedfkbA. fkbA encodes a component of the FK506 polyketide synthase, a complex enzyme system which catalyzes synthesis of the polyketide portion of FK506. The predicted product of genefkbA is a 630660-Da protein (6420 amino acids) that contains 1 9 independent domains with a high degree of amino acid sequence similarity to the catalytic activities of known fatty acid synthases. The identified domains are arranged into four repeated modules with a linear organization precisely as that of animal fatty acid synthase and type I polyketide synthase. Each module participates in one round of chain extension and subsequent processing and thus FkbA polypeptide catalyzes four of the ten condensation steps required for synthesis of the FK506 macrolactone ring. Disruption of jkbA results in the generation of an FK506 non-producing mutant demonstrating direct involvement of fkbA in the biosynthesis of FK506.

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6‐Deoxyerythronolide‐B synthase 2 from Saccharopolyspora erythraea

Cited by 170 publications

References 57 publications

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin‐producing polyketide synthase from Saccharopolyspora erythraea

Structural Organization of A Multifunctional Polyketide Synthase Involved in the Biosynthesis of the Macrolide Immunosuppressant FK506

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