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Information on EC 2.3.1.94 - 6-deoxyerythronolide-B synthase and Organism(s) Saccharopolyspora erythraea and UniProt Accession Q03132

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IUBMB Comments
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 . The polyketide product is synthesized by the processive action of a loading didomain, six extension modules and a terminal thioesterase domain . 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 .
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Saccharopolyspora erythraea
UNIPROT: Q03132
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The taxonomic range for the selected organisms is: Saccharopolyspora erythraea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
polyketide synthase, 6-deoxyerythronolide b, 6-deoxyerythronolide b synthase, debs1, erythromycin polyketide synthase, debs3, deoxyerythronolide b synthase, debs2, 6-deoxyerythronolide-b synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
deoxyerythronolide B synthase 2
-
polyketide synthase
-
6-deoxyerythronolide B
-
-
6-deoxyerythronolide B synthase
deoxyerythronolide B synthase
deoxyerythronolide B synthase 1
-
deoxyerythronolide B synthase 3
-
erythromycin polyketide synthase
-
erythronolide condensing enzyme
-
-
-
-
erythronolide synthase
-
polyketide synthase
synthase, erythronolide
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
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
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
propanoyl-CoA + 6 (2S)-methylmalonyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide B + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
-
-
-
?
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 + (2S)-methylmalonyl-CoA + NADPH
(3R,4S,5R,6R)-6-ethyl-4-hydroxy-3,5-dimethyloxan-2-one + ?
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
-
product of truncated enzyme consisting of loading didomain-module1-module2+thioesterase
-
?
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
-
product of truncated enzyme consisting of loading didomain-module1-module2-module3+thioesterase
-
?
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
-
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
-
?
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
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
propanoyl-CoA + 6 (2S)-methylmalonyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide B + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
-
-
-
?
6 methylmalonyl-CoA + propionyl-CoA + 6 NADPH + 6 H+
6-deoxyerythronolide b + 7 CoA + 6 CO2 + H2O + 6 NADP+
show the reaction diagram
-
formation of a key intermediate in the biosynthesis of erythromycin
-
?
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
?
-
-
unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase, ratchet mechanism, overview
-
-
?
INHIBITOR
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
Tetrahydrocerulenin
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0067 - 0.048
propanoyl-CoA
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
ketoreductases from DEBS modules 2 and 5 display little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. The ketoreductase from DEBS module 1 shows an about 10-50fold preference for substrate attached to its native ACP domain, whereas the ketoreductase from DEBS module 6 displays an about 10fold preference for the ACP from DEBS module 5
additional information
-
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
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
ERYA2_SACER
3567
0
374421
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
-
ACP domain
33000
thioesterase domain, 2 * 33000
37000
-
x * 37000, enzyme may be a component of a multifunctional protein or may exist as a dimer SDS-PAGE
66000
thioesterase domain, homodimer
75000
-
gel filtration
90000
-
crosslinked ketosynthase3-acyltransferase3-ACP3 and ketosynthase5-acyltransferase5-ACP5 domains, determined by SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 37000, enzyme may be a component of a multifunctional protein or may exist as a dimer SDS-PAGE
homodimer
thioesterase domain, 2 * 33000
oligomer
-
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
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C211A
mutation at the ketosynthase domain active site, exhibits no turnover activity, but is still a competent methylmalonyl-ACP decarboxylase
H346A
mutation at the ketosynthase domain, mutant exhibits reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction
H384A
mutation at the ketosynthase domain, residue contributes to methylmalonyl-ACP decarboxylation
H457A
activity similar to wild-type
K379A
mutation at the ketosynthase domain, residue promotes C-C bond formation
N381A
increase in activity
N455L
activity similar to wild-type
Q386E
about 25% of wild-type activity
S315A
mutation at the ketosynthase domain, residue plays a role in coupling decarboxylation to C-C bond formation
T209A
increase in activity
V187A/Y189R
mutant is able to utilize non-natural alkynyl-modified extender units
Y189R
mutant produces the propargyl analogue of 10-deoxymethynolide as the major product, while the wild-type enzyme prefers the natural substrate 2-methylmalonyl-CoA to provide the natural macrolactone 10-deoxymethynolide as the major product
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
susceptible to proteolysis
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
using a Ni-agarose and a Poros HQ column
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
a variant of DEBS1 is cloned into the vector pET21c
expression in Bacillus subtilis
expression in Escherichia coli
for expression in Escherichia coli cells
-
heterologous expression in Streptomyces coelicolor and Escherichia coli
-
the ACP construct is engineered with the FLAG epitope
-
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
the thioesterase domain from deoxyerythronolide B synthase is cloned for expression in Escherichia coli cells
the three DEBS genes are sequentially integrated into the Escherichia coli BAP1 chromosome
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
a derivative of Escherichia coli is genetically engineered to produce 6-deoxyerythronolide B, the macrocyclic core of the antibiotic erythromycin
analysis
kinetic assay for the megasynthase that stoichiometrically relates NADPH consumption to polyketide production
biotechnology
synthesis
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Roberts, G.; Leadly, P.F.
Use of [3H]tetrahydrocerulenin to assay condensing enzyme activity in Streptomyces erythreus
Biochem. Soc. Trans.
12
642-643
1984
Saccharopolyspora erythraea
-
Manually annotated by BRENDA team
Khosla, C.; Tang, Y.; Chen, A.Y.; Schnarr, N.A.; Cane, D.E.
Structure and mechanism of the 6-deoxyerythronolide B synthase
Annu. Rev. Biochem.
76
195-221
2007
Saccharopolyspora erythraea
Manually annotated by BRENDA team
Wong, F.T.; Chen, A.Y.; Cane, D.E.; Khosla, C.
Protein-protein recognition between acyltransferases and acyl carrier proteins in multimodular polyketide synthases
Biochemistry
49
95-102
2010
Saccharopolyspora erythraea (Q03131)
Manually annotated by BRENDA team
Kapur, S.; Worthington, A.; Tang, Y.; Cane, D.E.; Burkart, M.D.; Khosla, C.
Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase
Bioorg. Med. Chem. Lett.
18
3034-3038
2008
Saccharopolyspora erythraea
Manually annotated by BRENDA team
Pistorino, M.; Pfeifer, B.A.
Efficient experimental design and micro-scale medium enhancement of 6-deoxyerythronolide B production through Escherichia coli
Biotechnol. Prog.
25
1364-1371
2009
Saccharopolyspora erythraea
Manually annotated by BRENDA team
Wang, Y.; Pfeifer, B.
6-Deoxyerythronolide B production through chromosomal localization of the deoxyerythronolide B synthase genes in E. coli
Metab. Eng.
10
33-38
2008
Saccharopolyspora erythraea
Manually annotated by BRENDA team
Katz, L.
The DEBS paradigm for type I modular polyketide synthases and beyond
Methods Enzymol.
459
113-142
2009
Saccharopolyspora erythraea
Manually annotated by BRENDA team
Tsai, S.C.; Miercke, L.J.W.; Krucinski, J.; Gokhale, R.; Chen, J.C.H.; Foster, P.G.; Cane, D.E.; Khosla, C.; Stroud, R.M.
Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel
Proc. Natl. Acad. Sci. USA
98
14808-14813
2001
Saccharopolyspora erythraea (Q03133)
Manually annotated by BRENDA team
Pfeifer, B.A.; Admiraal, S.J.; Gramajo, H.; Cane, D.E.; Khosla, C.
Biosynthesis of complex polyketides in a metabolically engieered strain of Escherichia coli
Science
291
1790-1792
2001
Saccharopolyspora erythraea (Q03131), Saccharopolyspora erythraea (Q03132), Saccharopolyspora erythraea (Q03133), Saccharopolyspora erythraea
Manually annotated by BRENDA team
Charkoudian, L.K.; Liu, C.W.; Capone, S.; Kapur, S.; Cane, D.E.; Togni, A.; Seebach, D.; Khosla, C.
Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase
Protein Sci.
20
1244-1255
2011
Saccharopolyspora erythraea
Manually annotated by BRENDA team
Kumpfmueller, J.; Methling, K.; Fang, L.; Pfeifer, B.A.; Lalk, M.; Schweder, T.
Production of the polyketide 6-deoxyerythronolide B in the heterologous host Bacillus subtilis
Appl. Microbiol. Biotechnol.
100
1209-1220
2016
Saccharopolyspora erythraea (Q03131 and Q03132 and Q03133), Saccharopolyspora erythraea
Manually annotated by BRENDA team
Dunn, B.J.; Watts, K.R.; Robbins, T.; Cane, D.E.; Khosla, C.
Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases
Biochemistry
53
3796-3806
2014
Saccharopolyspora erythraea (Q03131 and Q03132 and Q03133)
Manually annotated by BRENDA team
Argyropoulos, P.; Bergeret, F.; Pardin, C.; Reimer, J.M.; Pinto, A.; Boddy, C.N.; Schmeing, T.M.
Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis
Biochim. Biophys. Acta
1860
486-497
2016
Saccharopolyspora erythraea (Q03133)
Manually annotated by BRENDA team
Lowry, B.; Robbins, T.; Weng, C.H.; OBrien, R.V.; Cane, D.E.; Khosla, C.
In vitro reconstitution and analysis of the 6-deoxyerythronolide B synthase
J. Am. Chem. Soc.
135
16809-16812
2013
Saccharopolyspora erythraea (Q03131 and Q03132 and Q03133)
Manually annotated by BRENDA team
Edwards, A.L.; Matsui, T.; Weiss, T.M.; Khosla, C.
Architectures of whole-module and bimodular proteins from the 6-deoxyerythronolide B synthase
J. Mol. Biol.
426
2229-2245
2014
Saccharopolyspora erythraea (Q03131 and Q03132 and Q03133), Saccharopolyspora erythraea
Manually annotated by BRENDA team
Koryakina, I.; Kasey, C.; McArthur, J.B.; Lowell, A.N.; Chemler, J.A.; Li, S.; Hansen, D.A.; Sherman, D.H.; Williams, G.J.
Inversion of extender unit selectivity in the erythromycin polyketide synthase by acyltransferase domain engineering
ACS Chem. Biol.
12
114-123
2017
Saccharopolyspora erythraea (Q03133)
Manually annotated by BRENDA team
Robbins, T.; Kapilivsky, J.; Cane, D.E.; Khosla, C.
Roles of conserved active site residues in the ketosynthase domain of an assembly line polyketide synthase
Biochemistry
55
4476-4484
2016
Saccharopolyspora erythraea (Q03131 and Q03132 and Q03133)
Manually annotated by BRENDA team
Ostrowski, M.P.; Cane, D.E.; Khosla, C.
Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases
J. Antibiot.
69
507-510
2016
Saccharopolyspora erythraea (Q03131 and Q03132 and Q03133)
Manually annotated by BRENDA team