4 research outputs found
Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis
Enediynes are potent natural product
anticancer antibiotics, and
are classified as 9- or 10-membered according to the size of their
enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores
are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase
(TE), and PKSE-associated enzymes. Although the divergence between
9- and 10-membered enediyne core biosynthesis remains unclear, it
has been observed that nascent polyketide intermediates, tethered
to the acyl carrier protein (ACP) domain of PKSE, could be released
by TE in the absence of the PKSE-associated enzymes. In this study,
we determined the crystal structure of SgcE10, the TE that participates
in the biosynthesis of the 9-membered enediyne C-1027. Structural
comparison of SgcE10 with CalE7 and DynE7, two TEs that participate
in the biosynthesis of the 10-membered enediynes calicheamicin and
dynemicin, respectively, revealed that they share a common α/β
hot-dog fold. The amino acids involved in both substrate binding and
catalysis are conserved among SgcE10, CalE7, and DynE7. The volume
and the shape of the substrate-binding channel and active site in
SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered
enediyne biosynthetic machineries bind the linear form of similar
ACP-tethered polyene intermediates. Taken together, these findings
further support the proposal that the divergence between 9- and 10-membered
enediyne core biosynthesis occurs beyond PKSE and TE catalysis
Structure of the <i>ent</i>-Copalyl Diphosphate Synthase PtmT2 from <i>Streptomyces platensis</i> CB00739, a Bacterial Type II Diterpene Synthase
Terpenoids are the
largest and most structurally diverse family
of natural products found in nature, yet their presence in bacteria
is underappreciated. The carbon skeletons of terpenoids are generated
through carbocation-dependent cyclization cascades catalyzed by terpene
synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate
ionization and protonation, respectively, and protein structures of
both types are known. Most plant diterpene synthases (DTSs) possess
three α-helical domains (αβγ), which are thought
to have arisen from the fusion of discrete, ancestral bacterial type
I TSs (α) and type II TSs (βγ). Type II DTSs of
bacterial origin, of which there are no structurally characterized
members, are a missing piece in the structural evolution of TSs. Here,
we report the first crystal structure of a type II DTS from bacteria.
PtmT2 from <i>Streptomyces platensis</i> CB00739 was verified
as an <i>ent</i>-copalyl diphosphate synthase involved in
the biosynthesis of platensimycin and platencin. The crystal structure
of PtmT2 was solved at a resolution of 1.80 Ã…, and docking studies
suggest the catalytically active conformation of geranylgeranyl diphosphate
(GGPP). Site-directed mutagenesis confirmed residues involved in binding
the diphosphate moiety of GGPP and identified DxxxxE as a potential
Mg<sup>2+</sup>-binding motif for type II DTSs of bacterial origin.
Finally, both the shape and physicochemical properties of the active
sites are responsible for determining specific catalytic outcomes
of TSs. The structure of PtmT2 fundamentally advances the knowledge
of bacterial TSs, their mechanisms, and their role in the evolution
of TSs
Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C‑1027
C-1027 is a chromoprotein
enediyne antitumor antibiotic, consisting
of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore
features a nine-membered enediyne core appended with three peripheral
moieties, including an (<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine.
In a convergent biosynthesis of the C-1027 chromophore, the (<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine moiety is appended
to the enediyne core by the free-standing condensation enzyme SgcC5.
Unlike canonical condensation domains from the modular nonribosomal
peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes
ester-bond formation, as demonstrated in vitro, between SgcC2-tethered
(<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine and (<i>R</i>)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core
as an acceptor substrate. Here, we report that (i) genes encoding
SgcC5 homologues are widespread among both experimentally confirmed
and bioinformatically predicted enediyne biosynthetic gene clusters,
forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar
overall structure with the canonical condensation domains but forms
a homodimer in solution, the active site of which is located in a
cavity rather than a tunnel typically seen in condensation domains,
and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl
group, while a hydrogen-bond network in SgcC5 prefers the <i>R</i>-enantiomer of the acceptor substrate, accounting for the
regio- and stereospecific ester-bond formation between SgcC2-tethered
(<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine and (<i>R</i>)-1-phenyl-1,2-ethanediol upon acid–base catalysis.
These findings expand the catalytic repertoire and reveal new insights
into the structure and mechanism of condensation enzymes
Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C‑1027 in <i>Streptomyces globisporus</i>
C-1027
is a chromoprotein enediyne antitumor antibiotic produced
by <i>Streptomyces globisporus</i>. In the last step of
biosynthesis of the (<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine
moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose
a two-component monooxygenase that hydroxylates the C-5 position of
(<i>S</i>)-3-chloro-β-tyrosine. This two-component
monooxygenase is remarkable for two reasons. (i) SgcE6 specifically
reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl
carrier protein (PCP)-tethered substrate. To address the molecular
details of substrate specificity, we determined the crystal structures
of SgcE6 and SgcC at 1.66 and 2.63 Ã… resolution, respectively.
SgcE6 shares a similar β-barrel fold with the class I HpaC-like
flavin reductases. A flexible loop near the active site of SgcE6 plays
a role in FAD binding, likely by providing sufficient space to accommodate
the AMP moiety of FAD, when compared to that of FMN-utilizing homologues.
SgcC shows structural similarity to a few other known FADH<sub>2</sub>-dependent monooxygenases and sheds light on some biochemically but
not structurally characterized homologues. The crystal structures
reported here provide insights into substrate specificity, and comparison
with homologues provides a catalytic mechanism of the two-component,
FADH<sub>2</sub>-dependent monooxygenase (SgcE6 and SgcC) that catalyzes
the hydroxylation of a PCP-tethered substrate