Information on EC 1.13.12.17 - dichloroarcyriaflavin A synthase

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The expected taxonomic range for this enzyme is: Streptomyces

EC NUMBER
COMMENTARY hide
1.13.12.17
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RECOMMENDED NAME
GeneOntology No.
dichloroarcyriaflavin A synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
dichlorochromopyrrolate + 4 O2 + 4 NADH + 4 H+ = dichloroarcyriaflavin A + 2 CO2 + 6 H2O + 4 NAD+
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
rebeccamycin biosynthesis
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Staurosporine biosynthesis
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
dichlorochromopyrrolate,NADH:oxygen 2,5-oxidoreductase (dichloroarcyriaflavin A-forming)
The conversion of dichlorochromopyrrolate to dichloroarcyriaflavin A is a complex process that involves two enzyme components. RebP is an NAD-dependent cytochrome P-450 oxygenase that performs an aryl-aryl bond formation yielding the six-ring indolocarbazole scaffold [1]. Along with RebC, a flavin-dependent hydroxylase, it also catalyses the oxidative decarboxylation of both carboxyl groups. The presence of RebC ensures that the only product is the rebeccamycin aglycone dichloroarcyriaflavin A [2]. The enzymes are similar, but not identical, to StaP and StaC, which are involved in the synthesis of staurosporine [3].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
TP-A0274
SwissProt
Manually annotated by BRENDA team
TP-A0274
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
dichlorochromopyrrolate + 4 O2 + 4 NADH + 4 H+
1,11-dichloroarcyriaflavin A + 2 CO2 + 6 H2O + 4 NAD+
show the reaction diagram
dichlorochromopyrrolate + 4 O2 + 4 NADH + 4 H+
dichloroarcyriaflavin A + 2 CO2 + 6 H2O + 4 NAD+
show the reaction diagram
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
dichlorochromopyrrolate + 4 O2 + 4 NADH + 4 H+
1,11-dichloroarcyriaflavin A + 2 CO2 + 6 H2O + 4 NAD+
show the reaction diagram
Q06IS2
the reaction is catalyzed by enzyme StaP. StaC and RebC acting to direct the level of oxidation in the newly formed five-membered ring. Biosynthesis of the antitumor indolocarbazoles rebeccamycin and staurosporine by streptomycetes
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dichlorochromopyrrolate + 4 O2 + 4 NADH + 4 H+
dichloroarcyriaflavin A + 2 CO2 + 6 H2O + 4 NAD+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome P450
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StaP is a cytochrome P450 enzyme
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Ferredoxin
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required for activity
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NADH
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NADH is the preferred reductant for StaC- and RebC-mediated turnover of FAD required for activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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StaP, a cytochrome P450 enzyme, catalyzes an aryl-aryl bond-forming reaction to give a six-ring indolocarbazole scaffold, as well as mediating decarboxylation and oxidation of the putative dicarboxypyrrole moiety. This action requires two to four cycles of net two-electron substrate oxidation at the catalytic heme center. StaP produces three distinct products, differing in oxidation level. For the production of K252c from chromopyrrolic acid, a net four-electron oxidation is required. The generation ofarcyriaflavin A from chromopyrrolic acid requires an overall eight-electron oxidation. StaP is thus unusual in the apparent lack of oxidative control it possesses over the outcome of its catalytic turnover. Control of the overall oxidation route is provided by a second enzyme StaC, which imparts the net effect of directing the oxidation level of the pyrrole-derived ring. While StaP in isolation gives three aglycone forms, StaP and StaC turn over chromopyrrolic acid to give only a single product, K252c. Similarly, RebC guides the turnover of chromopyrrolic acid toward the more highly oxidized maleimide-bearing aglycone, arcyriaflavin A
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 48000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
X-ray crystal structures of chromopyrrolic acid-bound and chromopyrrolic acid-free forms of enzyme component StaP. The enzyme attains a more ordered conformation upon binding of the substrate. Substantial conformational changes are observed at and around the substrate-binding pocket to optimize interactions with the enzyme. The enzyme–chromopyrrolic acid complex structure reveals that chromopyrrolic acid has a twisted-butterfly shape in the active site, which is perpendicular to the heme plane
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of StaP, StaC, and RebC in Escherichia coli
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the coding region for the enzyme component StaP is cloned into expression vector pET26b(+) (Novagen, San Diego, CA), with a C-terminal 6 * His tag, and overexpressed in Escherichia coli strain BL21
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H250A
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the amount of the catalytic product K252c decreases to 25%
H250F
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the amount of the catalytic product K252c decreases to 72%