6 research outputs found
Photoinduced Pd-Catalyzed Dearomative 2,5-Difunctionalizition of Furans via Cascade C–C/C–O Bond Formation
We report an efficient and mild approach for radical
dearomatization
via photoinduced palladium-catalyzed reaction of three components
(i.e., furans, alcohols, and bromoalkanes). In this strategy, various
functionalized spiro-heterocycles were prepared from furans in one
step via cascade C–C/C–O bond formation under redox
neutral conditions
Structure Reveals Regulatory Mechanisms of a MaoC-Like Hydratase from <i>Phytophthora capsici</i> Involved in Biosynthesis of Polyhydroxyalkanoates (PHAs)
<div><p>Background</p><p>Polyhydroxyalkanoates (PHAs) have attracted increasing attention as “green plastic” due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC), which belongs to (<i>R</i>)-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (<i>R</i>)-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic.</p> <p>Methodology/Principal Findings</p><p>We have determined the crystal structure of a new MaoC recognized from <i>Phytophthora capsici</i>. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (<i>R</i>)-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an <i>α</i>-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC.</p> <p>Conclusions/Significance</p><p>The data in our study reveal the regulatory mechanism of an (<i>R</i>)-hydratase, providing information on enzyme engineering to produce low cost PHAs.</p> </div
The active site of MaoC.
<div><p>(A) Superposition between MaoC and other similar structures. The structures used for the comparisons are colored as follows: MaoC, salmon; Hs-H2, blue; Ct-H2, lemon yellow. The major difference distinguishing MaoC from similar structures is the hypothetical inhibitory segment marked in magenta.</p>
<p>(B) Superposition of the catalytic triad from MaoC (salmon), Hs-H2 (blue) and Ct-H2 (lemon yellow). The carbons, nitrogens, and oxygens of MaoC, Hs-H2 and Ct-H2 are colored in salmon, blue and red; slate, blue and red; lemon yellow, blue and red, respectively.</p>
<p>(C) Mutations of the catalytic triad abolished the enzymatic activity of MaoC. The purified native MaoC and three mutants (upper), Enoyl-CoA hydratase activity for MaoC and its variants detected by crotonyl-CoA substrate (lower). The activities were assayed by determining the hydration of crotonyl-CoA substrate. One unit of activity was defined as the removal of 1 µmol of crotonyl-CoA per minute. The specific activity was defined as the activity of per milligram protein. The specific activities of the purified mutants were: D194N, 2.6 U/mg; H199Q, 1.7 U/mg; G217A, 17.4 U/mg, respectively. Mean of three independent experiments was considered.</p></div
Overall structure of MaoC.
<div><p>(A) Sequence alignment of MaoC and its similar eukaryotic hydratases. Sequences used for the alignment are MaoC-like hydratase from <i>Phytophthora capsici</i> (MaoC), the corresponding parts of hydratase 2 regions of human (Hs-H2) and 2-enoyl-CoA hydratase 2 part of <i>Candida tropicalis</i> Mfe2p (Ct-H2). The corresponding SwissProt identifiers of MaoC, Hs-H2 and Ct-H2 are E3T2G3, P51659 and P22414, respectively. The secondary structure elements of MaoC, α-helices (black lines) and β-strands (black arrows), are marked above the sequence. Black vertical arrows indicate the catalytic residues of MaoC. The black line below the sequence alignment indicates the hydratase 2 motif, and the magenta line above the sequence alignment shows the regions of the hypothetical inhibitory segment of MaoC (residues 63-88). Residues participating in substrate binding and dimerization are labeled with black and cyan circles, respectively.</p>
<p>(B) Different views of MaoC structure. The N- and C-domains, the intervening bridge, and the hypothetical inhibitory segment are colored in salmon, orange, white, and magenta, respectively. The two orientations of the structure are related by 90 ° rotations.</p></div
Dimerization is required for the enzyme activity of MaoC.
<div><p>(A) MaoC forms a homodimer in crystals. The key <i>α</i>-helices participating in dimerization (<i>α</i>2 and <i>α</i>2’, <i>α</i>6 and <i>α</i>6’, <i>α</i>3 and <i>α</i>3’, <i>α</i>7 and <i>α</i>7’) are labeled. The contacts of the central four-helix bundle are highlighted in a black frame.</p>
<p>(B) The detailed dimeric interactions of the central four-helical bundle. The carbons, nitrogens, and oxygens of the side chains from the two monomeric MaoC are colored in gray, blue and red (Chain A); yellow, blue and red (Chain B), respectively.</p>
<p>(C) MaoC forms dimer in solution. Shown in the figure are the gel filtration assay profiles of native and three indicated mutant MaoC proteins. The molecular weights of standard proteins are shown.</p>
<p>(D) Monomeric MaoC has no enzymatic activity. The assay is similar to that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080024#pone-0080024-g002" target="_blank">Figure 2C</a>.</p></div
An inhibitory segment of MaoC.
<div><p>(A) Superposition of the molecule A (salmon) over B (wheat) of MaoC shows the inhibitor segment (magenta) in the molecule B is mostly broken.</p>
<p>(B) The extra residues of MaoC (63-88aa) form a curved loop and make compact contacts with the <i>α</i>7.</p>
<p>(C) Deletion of the non-conserved segment of MaoC has no effect on its dimerization. The assay is similar to that show in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080024#pone-0080024-g003" target="_blank">Figure 3C</a>.</p>
<p>(D) Deletion of the non-conserved segment of MaoC enhances its activity. The assay is similar to that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080024#pone-0080024-g002" target="_blank">Figure 2C</a>.</p></div