Information on EC 2.3.1.94 - 6-deoxyerythronolide-B synthase

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The expected taxonomic range for this enzyme is: Saccharopolyspora erythraea

EC NUMBER
COMMENTARY hide
2.3.1.94
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RECOMMENDED NAME
GeneOntology No.
6-deoxyerythronolide-B synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
propanoyl-CoA + 6 (2S)-methylmalonyl-CoA + 6 NADPH + 6 H+ = 6-deoxyerythronolide B + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of 12-, 14- and 16-membered macrolides
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Biosynthesis of antibiotics
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erythromycin D biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
propanoyl-CoA:(2S)-methylmalonyl-CoA malonyltransferase (cyclizing)
The product, 6-deoxyerythronolide B, contains a 14-membered lactone ring and is an intermediate in the biosynthesis of erythromycin antibiotics. Biosynthesis of 6-deoxyerythronolide B requires 28 active sites that are precisely arranged along three large polypeptides, denoted DEBS1, -2 and -3 [6]. The polyketide product is synthesized by the processive action of a loading didomain, six extension modules and a terminal thioesterase domain [5]. Each extension module contains a minimum of a ketosynthase (KS), an acyltransferase (AT) and an acyl-carrier protein (ACP). The KS domain both accepts the growing polyketide chain from the previous module and catalyses the subsequent decarboxylative condensation between this substrate and an ACP-bound methylmalonyl extender unit, introduce by the AT domain. This combined effort gives rise to a new polyketide intermediate that has been extended by two carbon atoms [5].
CAS REGISTRY NUMBER
COMMENTARY hide
87683-77-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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enzyme-substrate interactions are involved in the translocation of a polyketide from ACP2 to ketosynthase KS3, the ACP domain of the 6-deoxyerythronolide B synthase contributes to its association with its ketosynthase, KS, translocation partner, models for KS-ACP recognition during chain elongation and chain translocation, docking model, overview. Determination of selective protein–protein interactions between the two partners using CF3-S-ACP as probe. The acyl chain substrate also has a significant influence on this interaction
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
6 methylmalonyl-CoA + propionyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide b + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
propanoyl-CoA + 5 (2S)-methylmalonyl-CoA + (2S)-ethylmalonyl-CoA + 6 NADPH + 6 H+
(3R,4S,5R,6R)-6-ethyl-4-hydroxy-3,5-dimethyltetrahydro-2H-pyran-2-one + ?
show the reaction diagram
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product of truncated enzyme consisting of loading didomain-module1-module2+thioesterase
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?
propanoyl-CoA + 5 (2S)-methylmalonyl-CoA + (2S)-ethylmalonyl-CoA + 6 NADPH + 6 H+
(3R,5R,6S)-6-[(2S,3R)-3-hydroxypentan-2-yl]-3,5-dimethyldihydro-2H-pyran-2,4(3H)-dione + ?
show the reaction diagram
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product of truncated enzyme consisting of loading didomain-module1-module2-module3+thioesterase
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?
propanoyl-CoA + 5 (2S)-methylmalonyl-CoA + (2S)-ethylmalonyl-CoA + 6 NADPH + 6 H+
8-ethyl-8-desmethyl-6-deoxyerythronolide B + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
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putative analog, 8-ethyl-8-desmethyl-6-deoxyerythronolide B, is generated by the assembly line in the presence of non-limiting concentrations of ethylmalonyl-CoA and is produced in comparable amounts to the natural 6-dEB product
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?
propanoyl-CoA + 6 (2S)-methylmalonyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide B + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
6 methylmalonyl-CoA + propionyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide b + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
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formation of a key intermediate in the biosynthesis of erythromycin
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?
propanoyl-CoA + 6 (2S)-methylmalonyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide B + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
additional information
?
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unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase, ratchet mechanism, overview
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3R,5R)-diphenyl 6-(3,5-dihydroxy-6-phenylhexanoylamino)hexylphosphonate
phosphonate ester, close structural homolog of substrates known to undergo macrocyclization and hydrolysis. The ester effectively inhibits the enzyme, but does not lead to co-complex structures. Pretreatment of recombinant thioesterase, residues15-283, with inhibitor for 18 h abolishes enzymatic activity for hydrolysis of the N-acetyl-cysteamine thioester of 3-hydroxyheptanoate
(3S,5R)-diphenyl 6-(3,5-dihydroxy-6-phenylhexanoylamino)hexylphosphonate
phosphonate ester, close structural homolog of substrates known to undergo macrocyclization and hydrolysis. The ester effectively inhibits the enzyme, but does not lead to co-complex structures. Pretreatment of recombinant thioesterase, residues15-283, with inhibitor for 18 h abolishes enzymatic activity for hydrolysis of the N-acetyl-cysteamine thioester of 3-hydroxyheptanoate
prop-2-en-1-ylphosphonic acid
88% inhibition
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Tetrahydrocerulenin
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
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ACP domain
33000
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thioesterase domain, 2 * 33000
37000
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x * 37000, enzyme may be a component of a multifunctional protein or may exist as a dimer SDS-PAGE
66000
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thioesterase domain, homodimer
75000
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gel filtration
90000
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crosslinked ketosynthase3-acyltransferase3-ACP3 and ketosynthase5-acyltransferase5-ACP5 domains, determined by SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 37000, enzyme may be a component of a multifunctional protein or may exist as a dimer SDS-PAGE
homodimer
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thioesterase domain, 2 * 33000
oligomer
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the polyketide product is synthesized by the processive action of a loading didomain, six extension modules and a terminal thioesterase domain, as a minimal requirement for chain extension, each module contains a set of three functional domains, a ketosynthase, an acyltransferae, and an acyl carrier protein
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
small-angle X-ray scattering analyses of a whole module and a bimodule from 6-deoxyerythronolide B synthase DEBS, as well as a set of domains. Data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the modules 5 and 6 stack collinearly along the 2fold axis of symmetry
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structures of a construct of DEBS thioesterase domain alone at 1.7 A, and DEBS thioesterase domain bound with a simple allylphosphonate, prop-2-en-1-ylphosphonic acid, at 2.1 A resolution
the crystal structure of the thioesterase domain from deoxyerythronolide B synthase is solved to 2.8 A resolution
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
susceptible to proteolysis
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
using a Ni-agarose and a Poros HQ column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a variant of DEBS1 is cloned into the vector pET21c; into the vector pET21c; into the vector pET21c
expression in Bacillus subtilis
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expression in Escherichia coli
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for expression in Escherichia coli cells
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heterologous expression in Streptomyces coelicolor and Escherichia coli
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the ACP construct is engineered with the FLAG epitope
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the acyltransferase domains, AT1, AT2, AT4, AT5 and AT6, from DEBS are expressed as stand-alone proteins with their ketosynthase-to-acyltransferase and post-acyltransferase linkers, ketosynthase3-acyltransferase3 and ketosynthase6-acyltransferase6 didomains and discrete acyl carrier protein domain expression vectors are constructed, the vector pET28a is used for expression in Escherichia coli BL21DE3 cells
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the thioesterase domain from deoxyerythronolide B synthase is cloned for expression in Escherichia coli cells
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the three DEBS genes are sequentially integrated into the Escherichia coli BAP1 chromosome
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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kinetic assay for the megasynthase that stoichiometrically relates NADPH consumption to polyketide production
biotechnology
synthesis
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the deoxyerythronolide B synthase genes are functionally expressed in Bacillus subtilis when the native eryAI–III operon is separated into three individual expression cassettes with optimized ribosomal binding sites. A synthesis of 6-deoxyerythronolide B can be detected by using the acetoininducible acoA promoter and a fed-batch simulating EnBase-cultivation strategy. Bacillus subtilis is capable of the secretion of 6-deoxyerythronolide B into the medium. The deletion of the prpBD operon of Bacillus subtilis, responsible for propionyl-CoA utilization, results in a significant increase of the 6-deoxyerythronolide B product yield when exogenous propionate is provided
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