9 research outputs found
Diagrams Showing Regions of Flexibility in Truncated DapE Proteins.
<p>A) MOLMOL diagram of [ZnZn(<i>Vc</i>DapE<sup>T</sup>)] molecular dynamics. B) MOLMOL diagram of [ZnZn(<i>Hi</i>DapE<sup>T</sup>)] molecular dynamics (the thickness of the line is proportional to the variation of the protein structure during the simulation). The crystallographic temperature factors indicating that the most dynamic (in red) and the most rigid (in blue) parts of the protein: C) [ZnZn(<i>Vc</i>DapE<sup>T</sup>)]. D) [ZnZn(<i>Hi</i>DapE<sup>T</sup>)].</p
Dynamic light scattering data.
<p><i>R</i><sub>H</sub> ā hydrodynamic radius; MW ā molecular weight; Pd ā the polydispersity, or width of the distribution, in nm, determined using a cumulants analysis (where the data are fit to an assumed distribution of particle sizes and the average radius and spread of radii is reported); %Pd ā defined as Pd/<i>R</i><sub>H</sub>, the polydispersity divided by the estimated hydrodynamic radius from the cumulants fit of the autocorrelation function multiplied by 100; Baseline ā The measured value of the normalized intensity autocorrelation curve. Values of 1.000 indicate that the measured correlation curve has returned to the baseline within the defined time. Deviations from the theoretical value of 1.000 typically indicate a noisy baseline; SOS error- the sum of squares difference between the measured correlation curve and the best fit curve calculated using the cumulants method of analysis, where a dust and noise free monomodal (single distribution) low polydispersity (narrow distribution) sample is assumed.</p
The Role of the Dimerization Domain in the Stabilization of Loop V in WT-<i>Hi</i>DapE.
<p>A) Superimposition of the WT-<i>Hi</i>DapE (gray) and <i>Vc</i>DapE<sup>T</sup> (cyan) structures is shown. Loop V of WT-<i>Hi</i>DapE and <i>Vc</i>DapE<sup>T</sup> is labeled as <i>Hi</i>LV and <i>Vc</i>LV, respectively. WT-<i>Hi</i>DapE residues interacting with the sulfate ion (stick model) are shown as gray lines. Corresponding residues in <i>Vc</i>DapE<sup>T</sup> (except for R258 that is absent in the deletion mutant) are shown as yellow (R179 and R180) and orange (G214) lines. B) Specific orientation of the active site loop V in <i>Vc</i>DapE<sup>T</sup> and the corresponding loop in AAP. Overlay of the <i>Vc</i>DapE<sup>T</sup> (cyan) and AAP (purple) structures is shown. The AAP loop and <i>Vc</i>DapE<sup>T</sup> loop V are labeled as <i>Ap</i>LV and <i>Vc</i>LV, respectively. Stabilization of loop V in AAP by a disulfide bridge is indicated where Cys223 and Cys227 of AAP and the residues involved in zinc-binding in <i>Vc</i>DapE<sup>T</sup> are shown as sticks. Zinc ions of <i>Vc</i>DapE<sup>T</sup> are shown as black spheres. Zinc-bound ethylene glycol was omitted for clarity.</p
Molecular Dynamic Simulation Showing Regions of Flexibility in Catalytic Domain.
<p>A) [ZnZn(<i>Vc</i>DapE)]. B) [ZnZn(<i>Hi</i>DapE)]. C) AAP. The thickness of the line is proportional to the variation of the protein structure during the simulation. AS indicates the active site area, LVeq. equivalent of the LV loop in <i>Hi</i>DapE).</p
The Active Site of WT-<i>Hi</i>DapE Showing Loop V.
<p>T325 resides on loop V directly over the dinuclear active site.</p
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 Ī±/Ī²
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
Data and Refinement Statistics.
a<p><i>R</i><sub>merge</sub>ā=āĪ£<i><sub>hkl</sub></i>Ī£<i><sub>i</sub></i>|<i>Ii</i><sub>(</sub><i>hkl</i><sub>)</sub>āā©<i>I<sub>hkl</sub></i>āŖ|/Ī£<i><sub>hkl</sub></i>Ī£<i><sub>i</sub>I<sub>i</sub></i><sub>(<i>hkl</i>)</sub>, where I<i>i</i><sub>(</sub><i>hkl</i><sub>)</sub> is the <i>i</i>th observation of reflection <i>hkl</i>, and ā©<i>I<sub>hkl</sub></i>āŖ is the weighted average intensity for all observations <i>i</i> of reflection <i>hkl</i>.</p><p>, and .</p>b<p>Numbers in parentheses are values for the highest-resolution bin.</p>c<p>As defined by MOLPROBITY (M.F. āthe most favored/A.A additionally allowed).</p
Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic Cā1027
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-Ī²-tyrosine.
In a convergent biosynthesis of the C-1027 chromophore, the (<i>S</i>)-3-chloro-5-hydroxy-Ī²-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-Ī²-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-Ī²-tyrosine and (<i>R</i>)-1-phenyl-1,2-ethanediol upon acidābase catalysis.
These findings expand the catalytic repertoire and reveal new insights
into the structure and mechanism of condensation enzymes
<i>Bacillus anthracis</i> Inosine 5ā²-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ionā, Substrateā, and Product-Bound Complexes
Inosine 5ā²-monophosphate dehydrogenase (IMPDH)
catalyzes
the first unique step of the GMP branch of the purine nucleotide biosynthetic
pathway. This enzyme is found in organisms of all three kingdoms.
IMPDH inhibitors have broad clinical applications in cancer treatment,
as antiviral drugs and as immunosuppressants, and have also displayed
antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed
āapoā) form and in complex with its substrate, inosine
5ā²-monophosphate (IMP), and product, xanthosine 5ā²-monophosphate
(XMP). This is the first example of a bacterial IMPDH in more than
one state from the same organism. Furthermore, for the first time
for a prokaryotic enzyme, the entire active site flap, containing
the conserved Arg-Tyr dyad, is clearly visible in the structure of
the apoenzyme. Kinetic parameters for the enzymatic reaction were
also determined, and the inhibitory effect of XMP and mycophenolic
acid (MPA) has been studied. In addition, the inhibitory potential
of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors
was examined for the <i>B. anthracis</i> enzyme and compared
with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active
site and design of inhibitors that specifically target <i>B.
anthracis</i> and other microbial IMPDH enzymes