67 research outputs found
Hydrophobic tunnel.
<p><b>A.</b> Fluconazole in orientation 2 bound to CYP51<sub>Tc</sub>. Fluorinated edge of the 2,4-difluorophenyl ring faces heme macrocycle. Fluconazole and heme are shown as van der Waals spheres; residues within 7 Ã… of fluconazole as sticks. Color scheme for heme and fluconazole as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd-0000651-g003" target="_blank"><b>Fig. 3</b></a>. F105 superimposed from CYP51<sub>Tb</sub> is shown as semitransparent pink spheres. <b>B.</b> Front view of CYP51<sub>Tc</sub> clipped by plane (cyan) through substrate binding tunnel. Hydrophobic areas are orange, hydrophilic areas blue. Heme at end of tunnel: with van der Waals spheres in red. Fluconazole is removed for clarity. Image was prepared using CHIMERA <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd.0000651-Pettersen1" target="_blank">[60]</a>.</p
Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway
Cytochrome P450 monooxygenases
(P450s) are some of nature’s
most ubiquitous and versatile enzymes for performing oxidative metabolic
transformations. Their unmatched ability to selectively functionalize
inert C–H bonds has led to their increasing employment in academic
and industrial settings for the production of fine and commodity chemicals.
Many of the most interesting and potentially biocatalytically useful
P450s come from microorganisms, where they catalyze key tailoring
reactions in natural product biosynthetic pathways. While most of
these enzymes act on structurally complex pathway intermediates with
high selectivity, they often exhibit narrow substrate scope, thus
limiting their broader application. In the present study, we investigated
the reactivity of the P450 MycCI from the mycinamicin biosynthetic
pathway toward a variety of macrocyclic compounds and discovered that
the enzyme exhibits appreciable activity on several 16-membered ring
macrolactones independent of their glycosylation state. These results
were corroborated by performing equilibrium substrate binding experiments,
steady-state kinetics studies, and X-ray crystallographic analysis
of MycCI bound to its native substrate mycinamicin VIII. We also characterized
TylHI, a homologous P450 from the tylosin pathway, and showed that
its substrate scope is severely restricted compared to MycCI. Thus,
the ability of the latter to hydroxylate both macrocyclic aglycones
and macrolides sets it apart from related biosynthetic P450s and highlights
its potential for developing novel P450 biocatalysts with broad substrate
scope and high regioselectivity
Recommended from our members
Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition
Non-ribosomal peptides play a critical role in the clinic
as therapeutic
agents. To access more chemically diverse therapeutics, non-ribosomal
peptide synthetases (NRPSs) have been targeted for engineering through
combinatorial biosynthesis; however, this has been met with limited
success in part due to the lack of proper protein–protein interactions
between non-cognate proteins. Herein, we report our use of chemical
biology to enable X-ray crystallography, molecular dynamics (MD) simulations,
and biochemical studies to elucidate binding specificities between
peptidyl carrier proteins (PCPs) and adenylation (A) domains. Specifically,
we determined X-ray crystal structures of a type II PCP crosslinked
to its cognate A domain, PigG and PigI, and of PigG crosslinked to
a non-cognate PigI homologue, PltF. The crosslinked PCP-A domain structures
possess large protein–protein interfaces that predominantly
feature hydrophobic interactions, with specific electrostatic interactions
that orient the substrate for active site delivery. MD simulations
of the PCP-A domain complexes and unbound PCP structures provide a
dynamical evaluation of the transient interactions formed at PCP-A
domain interfaces, which confirm the previously hypothesized role
of a PCP loop as a crucial recognition element. Finally, we demonstrate
that the interfacial interactions at the PCP loop 1 region can be
modified to control PCP binding specificity through gain-of-function
mutations. This work suggests that loop conformational preferences
and dynamism account for improved shape complementary in the PCP-A
domain interactions. Ultimately, these studies show how crystallographic,
biochemical, and computational methods can be used to rationally re-engineer
NRPSs for non-cognate interactions
Overall structures of CYP51.
<p><b>A, B.</b> Fluconazole-bound CYP51<sub>Tc</sub> with selected α-helices labeled (PDB ID Code 2WUZ). <b>A.</b> Distal protein surface with respect to heme. <b>B.</b> Image is rotated ∼90° toward viewer. Protein backbone is depicted by cyan ribbon with the I-helix highlighted in magenta, BC-region in green, FG-region in blue. Heme (orange) and fluconazole are depicted by spheres. Fluconazole color scheme: carbon yellow, oxygen red, nitrogen blue, fluorine cyan. Images were prepared using PYMOL <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd.0000651-DeLano1" target="_blank">[58]</a> unless indicated otherwise. <b>C, D.</b> CYP51<sub>Tb</sub> in surface representation with the hydrophobic residues colored in golden yellow. In <b>C</b>, posaconazole is omitted for clarity, heme prosthetic group (pink) shows through the hydrophobic tunnel entrance. In <b>D</b>, two overlaid posaconazole conformers are shown protruding out of the tunnel opening. Bent conformer (chain B) is in red, extended conformer (chain D) is in blue. Images were generated using VMD program <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd.0000651-Humphrey1" target="_blank">[59]</a>.</p
Fe anomalous dispersion data collection and phasing statistics.
1<p>Values in parentheses are for highest-resolution shell; R<sub>sym</sub> is meaningless when the individual spot I/σI value is below 1.</p
Sequence alignments between host and pathogen CYP51.
<p>Sequence alignments between CYP51 from <i>Trypanosoma cruzi</i>, <i>Trypanosoma brucei, Aspergillus fumigatus</i>, <i>Candida albicans</i> and human. Accession numbers of the proteins in the Swiss-Prot/TrEMBL (<a href="http://us.expasy.org/sprot" target="_blank">http://us.expasy.org/sprot</a>) and NCBI (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>) databases are given next to the name of the protein. Alignments were performed using CLUSTALW program online <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd.0000651-Thompson1" target="_blank">[61]</a>. The figure was generated using ESPript <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd.0000651-Gouet1" target="_blank">[62]</a>. The secondary structure annotation and residue numbering at the top correspond to CYP51<sub>Tc</sub>, residue numbering at the bottom corresponds to human CYP51. The α-helices are labeled with capital letters according to generally accepted P450 nomenclature. The β-strands of large β-sheets are labeled with dashed numbers. Sequential numbers are used to label short two-residue β-strands. Residues within 7 Å of fluconazole are labeled with blue triangles. Additional residues constituting the hydrophobic tunnel are labeled with green triangles. Human H236 and H489 and the corresponding residues in the pathogenic species are highlighted in yellow. Residues corresponding to CYP51<sub>Tc</sub> I105 are highlighted in cyan. Mutation hot spots at the tunnel opening are marked with black stars. Gray stars highlight residues in alternate conformations.</p
Design and analysis of the expression vectors.
<p>Highlighted in bold are the constructs which led to the corresponding x-ray structures.</p
Comparison between CYP51 from different phyla.
<p><b>A.</b> CYP51<sub>Tc</sub> (cyan in <b>A</b>, <b>B</b> and <b>C</b>, PDB ID Code 2WUZ) and CYP51<sub>Tb</sub> (wheat, PDB ID Code 2WV2) superimposed with r.m.s.d. of 0.89 Å. Helices are represented by labeled cylinders. Fluconazole is omitted for clarity. <b>B.</b> CYP51<sub>Tc</sub> and CYP51<sub>Mt</sub> (golden yellow, PDB ID Code 2VKU) superimposed with r.m.s.d of 1.83 Å. <b>C.</b> CYP51<sub>Tc</sub> and CYP51<sub>h</sub> (lemon green, PDB ID Code: 3I3K) superimposed with r.m.s.d of 1.45 Å. In each panel, distal surface is shown on the right. Image on the left is rotated ∼90° toward viewer.</p
Spectral characterization of CYP51<sub>Tc</sub> and CYP51<sub>Tb</sub>.
<p>Soret and visible regions of the CYP51<sub>Tc</sub> (<b>A</b>) and CYP51<sub>Tb</sub> (<b>B</b>) spectra are shown. The ferric protein (dashed trace) was reduced with sodium dithionite to a ferrous form (solid trace) in the presence of CO. The spectra were recorded at room temperature in a 1 ml quartz cuvette containing 1 µM CYP51 in 10 mM Tris-HCl, pH 7.5, and 10% glycerol using a Cary UV-visible scanning spectrophotometer (Varian). CYP51<sub>Tc</sub> has a Soret maximum at 420 nm which upon reduction with sodium dithionite and CO binding shifts to 449 nm (<b>A</b>). CYP51<sub>Tb</sub> has a Soret maximum at 417 nm which upon reduction and CO binding shifts to 446 nm (<b>B</b>).</p
Fluconazole binding in CYP51<sub>Tc</sub>.
<p><b>A, B.</b> Stereoscopic view of CYP51<sub>Tc</sub> with fluconazole bound in active site. Side chains of the residues within 4 Ã… of fluconazole are in green. For clarity, A287, A291 and T295 are omitted. Main chain atoms are shown for M360. Fluconazole color scheme as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000651#pntd-0000651-g003" target="_blank"><b>Fig. 3</b></a>. Fragments of 2F<sub>o</sub>-F<sub>c</sub> electron density map calculated with the fluconazole coordinates omitted from the input file are shown as grey wire mesh. Chain A has been used in both structures to generate the images. <b>A.</b> Fluconazole orientation 1 in 2WX2 structure; OH-group of Y103 H-bonds to the amide nitrogen of M360. <b>B.</b> Fluconazole orientation 2 in the 2WUZ structure; peak in the positive F<sub>o</sub>-F<sub>c</sub> map (pink mesh) calculated with 2,4-difluorophenyl ring in flipped orientation superimposes with the 2-fluorine H-bonding to Y103.</p
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