Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase

Gokhale, Rajesh S.; Hunziker, Daniel; Cane, David E.; Khosla, Chaitan

doi:10.1016/s1074-5521(99)80008-8

Cited by 154 publications

(160 citation statements)

References 29 publications

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“…It has been speculated that these domains encode a thioesterase which functions to edit the product or purge synthetases of aberrant materials that might otherwise block the synthesis of normal products (6). Alternatively, in polyketide synthesis, thioesterases may be responsible for the release and concomitant cyclization of the processed chain (16). While the homology search described in this paper produced only a distant similarity with thioesterases, the correlation of the insertional inactivation of dltD with the defective hydrolytic cleavage of D-alanyl-ACP strongly supports the conclusion that DltD catalyzes thioesterase activity.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

2000

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(10) cloned, sequenced, and expressed the gene encoding this novel carrier protein, a homologue of the acyl carrier protein (ACP) involved in fatty acid biosynthesis. In addition to the genes encoding Dcl (DltA) and Dcp (DltC), the dlt operon contains two additional genes, dltB and dltD, encoding putative membrane proteins (10, 33).The goal of this paper is to establish the function of the Lactobacillus rhamnosus protein encoded by dltD. To accomplish this goal, the gene was cloned, sequenced, and expressed in Escherichia coli with and without the sequence encoding the N-terminal hydrophobic domain. Additionally, the gene was inactivated and its protein product was examined for thioesterase activity and carrier protein binding specificity. The results support a role for this protein in hydrolyzing mischarged D-alanyl-ACP and as a facilitator of D-alanine ligation to the carrier protein Dcp. MATERIALS AND METHODSBacterial strains, bacteriophages, and plasmids. All bacterial strains, phages, and plasmids used in this study are listed in Table 1. Chemicals and reagents. [ 35 S]dATP (600 Ci/mmol in 5 mM Tris-HCl [pH 7.5]), D-[14 C]alanine (43 mCi/mmol), and dithiothreitol (DTT) were products of ICN Biochemicals, Inc. Medium supplies were obtained from Difco Laboratories. Tetracycline, ampicillin, carbenicillin, erythromycin, EDTA, Tris, bis-Tris, cetyltrimethylammonium bromide (CTAB), and chlorhexidine were obtained from Sigma Chemical Co. Solutions of phenol-chloroform were products of Amresco Inc. Metricel filter membranes (GN-6) and Econo-Safe scintillation cocktail were purchased from Gelman Sciences and RPI Corp., respectively. Restriction enzymes were obtained from a number of suppliers and were used according to the manufacturer's instructions. Isopropyl-␤-D-galactoside (IPTG) and EcoRI (NotI) adapters were purchased from Gibco-BRL. The Genius System nonradioactive DNA labeling kit and DIG nucleic acid detection kit were purchased from Boehringer Mannheim GmbH. Gigapack II Plus packaging extracts, T4 DNA ligase, and PfuTurbo DNA polymerase were obtained from Stratagene. The Sequenase and PCR kits were purchased from United States Biochemical Corp. and MBI Fermentas, respectively. BioBlot nitrocellulose blotting membranes were purchased from Costar, Inc. The QIAprep spin miniprep, QIAquick nucleotide removal, and QIAquick gel extraction kits were obtained from Qiagen. Oligonucleotides were synthesized by Integrated DNA Technologies, Inc.Growth of bacteria. E. coli was cultured in either Luria-Bertrani (LB) broth or 2ϫ YT (2ϫ yeast extract-tryptone) medium and plated on either LB agar or LB agarose (41). L. rhamnosus 7469 and Lactobacillus casei 102S were cultured in MRS medium (Difco). For electroporation, L. casei 102S was grown in a low-salt medium (LL) containing (grams/liter) tryptone, 15; yeast extract, 5; MgSO 4 , 0.01; MnSO 4 , 0.005; glucose, 10; and 5 mM potassium phosphate buffer (pH 7.0). Overnight cultures were diluted 1:30 with fresh LL medium, and the cells were grown for an additional 3 to 3.5 ...

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

2000

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“…The products of such genes are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chainterminating thioesterase (TE I) domains (Gokhale et al, 1999). In fatty acid synthase complexes, TE IIs are alternative chain-terminating enzymes exhibiting hydrolase activity towards medium-chain-length acyl thioesters (Smith, 1994).…”

Section: Abbreviations : Pks Polyketide Synthase ; Te Thioesterasementioning

confidence: 99%

Type II thioesterase from Streptomyces coelicolor A3(2) The GenBank accession number for the sequence reported in this paper is AF109727.

et al. 2002

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Type I polyketide synthases (PKSs) are complexes of large, multimodular enzymes that catalyse biosynthesis of polyketide compounds via repetitive reaction sequences, during which each step is catalysed by a separate enzymic domain. Many type I PKSs, and also non-ribosomal peptide synthetase clusters, contain additional thioesterase genes located adjacent to PKS genes. These are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chain-terminating thioesterase (TE I) domains that are usually fused to the terminal PKS module. A gene of a new TE II, scoT, associated with the cluster of putative type I PKS genes from Streptomyces coelicolor A3(2), was found. The deduced amino acid sequence of the gene product shows extensive similarity to other authentic thioesterase enzymes, including conservation of characteristic motifs and residues involved in catalysis. When expressed in the heterologous host Streptomyces fradiae, scoT successfully complemented the resident TE II gene (tylO), and, by restoring a significant level of macrolide production, proved to be catalytically equivalent to the TylO protein. S 1 nuclease mapping of scoT revealed a single potential transcription start point with expression being switched on for a short period of time during a transition phase of growth.

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“…A type I TE domain is usually found at the carboxyl terminus of the last module to act in the sequence of events catalysed by a PKS or NRPS, whereas the TEII enzymes are separate, single proteins. The TEI is responsible for release of the acyl chain from the PKS (Gokhale et al, 1999), NRPS (Kohli et al, 2001;Schwarzer et al, 2001) or NRPS/PKS hybrid (Tang et al, 2000), whereas TEII is thought to serve an editing function to remove aberrant intermediates from the PKS and NRPS systems (Schwarzer et al, 2002). TEII loss-of-function mutations in some bacterial PKS, NRPS and NRPS/PKS gene clusters have been reported to result in greatly reduced polyketide or oligopeptide production (Butler et al, 1999;Doi-Katayama et al, 2000;Schneider & Marahiel, 1998;Xue et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…Plasmids pKOS207-129 and pBP130 expressing the DEBS1, and DEBS2 and DEBS3 proteins, respectively, from T7 promoters have been described previously (Murli et al, 2003;Pfeifer et al, 2001). Plasmid pKOS207-142a is similar to pKOS207-129 except that the NdeI-SpeI fragment encoding the DEBS1 PKS in pKOS207-129 is replaced by the NdeI-SpeI fragment encoding the DEBS1 module 2 only from pRSG64 (Gokhale et al, 1999). To generate an E. coli expression vector for ery-ORF5 that was compatible with the DEBS plasmids, the ery-ORF5 PCR fragment used to generate pKOS146-124B described below was cloned as a blunt PCR fragment into pCR-Blunt (Invitrogen), generating pKOS146-124, and sequenced.…”

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confidence: 99%

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

Hu¹,

Pfeifer

Chao

et al. 2003

Microbiology

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Bacterial modular polyketide synthase (PKS) genes are commonly associated with another gene that encodes a thioesterase II (TEII) believed to remove aberrantly loaded substrates from the PKS. Co-expression of the Saccharopolyspora erythraea ery-ORF5 TEII and eryA genes encoding 6-deoxyerythronolide B synthase (DEBS) in Streptomyces hosts eliminated or significantly lowered production of 8,89-deoxyoleandolide [15-nor-6-deoxyerythronolide B (15-nor-6dEB)], which arises from an acetate instead of a propionate starter unit. Disruption of the TEII gene in an industrial Sac. erythraea strain caused a notable amount of 15-norerythromycins to be produced by utilization of an acetate instead of a propionate starter unit and also resulted in moderately lowered production of erythromycin compared with the amount produced by the parental strain. A similar behaviour of the TEII gene was observed in Escherichia coli strains that produce 6dEB and 15-methyl-6dEB. Direct biochemical analysis showed that the ery-ORF5 TEII enzyme favours hydrolysis of acetyl groups bound to the loading acyl carrier protein domain (ACP L ) of DEBS. These results point to a clear role of the TEII enzyme, i.e. removal of a specific type of acyl group from the ACP L domain of the DEBS1 loading module.

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Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase

Cited by 154 publications

References 29 publications

Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

Type II thioesterase from Streptomyces coelicolor A3(2) The GenBank accession number for the sequence reported in this paper is AF109727.

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

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