49 research outputs found
Crystallographic Study Of The Phosphoethanolamine Transferase EptC required For Polymyxin Resistance And Motility In Campylobacter jejuni
The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 angstrom. cEptC adopts the alpha/beta/alpha fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN-enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.National Institutes of Health (grants AI064184, AI076322, GM106112Army Research Office (grantW911NF-12-1-0390)College of Natural SciencesOffice of the Executive Vice President and ProvostInstitute for Cellular and Molecular Biology at the University of Texas at AustinUS DOE DE-AC02-06CH11357National Institute of General Medical SciencesHoward Hughes Medical InstituteOffice of Science, Office of Basic Energy Sciences of the US Department of Energy DE-AC02-05CH11231Maria
Person and the Proteomics Facility at the University of Texas at Austin ES007784 (CRED) and
RP110782 (CPRIT)Molecular Bioscience
Employing Modular Polyketide Synthase Ketoreductases as Biocatalysts in the Preparative Chemoenzymatic Syntheses of Diketide Chiral Building Blocks
SummaryChiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, β-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100 mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale
Catalysis, Specificity, and ACP Docking Site of Streptomyces coelicolor Malonyl-CoA:ACP Transacylase
Structural Studies of an A2-Type Modular Polyketide Synthase Ketoreductase Reveal Features Controlling α‑Substituent Stereochemistry
Modular polyketide synthase ketoreductases
often set two stereocenters
when reducing intermediates in the biosynthesis of a complex polyketide.
Here we report the 2.55-Ã… resolution structure of an A2-type
ketoreductase from the 11th module of the amphotericin polyketide
synthase that sets a combination of l-α-methyl and l-β-hydroxyl stereochemistries and represents the final
catalytically competent ketoreductase type to be structurally elucidated.
Through structure-guided mutagenesis a double mutant of an A1-type
ketoreductase was generated that functions as an A2-type ketoreductase
on a diketide substrate analogue, setting an α-alkyl substituent
in an l-orientation rather than in the d-orientation
set by the unmutated ketoreductase. When the activity of the double
mutant was examined in the context of an engineered triketide lactone
synthase, the anticipated triketide lactone was not produced even
though the ketoreductase-containing module still reduced the diketide
substrate analogue as expected. These findings suggest that re-engineered
ketoreductases may be catalytically outcompeted within engineered
polyketide synthase assembly lines
Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG
Spinosyns A and D (spinosad), like many other complex
polyketides,
are tailored near the end of their biosyntheses through the addition
of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also
capable of adding other monosaccharides to the spinosyn aglycone (AGL)
from TDP-sugars; however, the substitution of UDP-d-glucose
for TDP-d-glucose as the donor substrate is known to result
in a >60000-fold reduction in <i>k</i><sub>cat</sub>.
Here,
we report the structure of SpnG at 1.65 Ã… resolution, SpnG bound
to TDP at 1.86 Ã… resolution, and SpnG bound to AGL at 1.70 Ã…
resolution. The SpnG–TDP complex reveals how SpnG employs N202
to discriminate between TDP- and UDP-sugars. A conformational change
of several residues in the active site is promoted by the binding
of TDP. The SpnG–AGL complex shows that the binding of AGL
is mediated via hydrophobic interactions and that H13, the potential
catalytic base, is within 3 Ã… of the nucleophilic 9-OH group
of AGL. A model for the Michaelis complex was constructed to reveal
the features that allow SpnG to transfer diverse sugars; it also revealed
that the rhamnosyl moiety is in a skew-boat conformation during the
transfer reaction
Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG
Spinosyns A and D (spinosad), like many other complex
polyketides,
are tailored near the end of their biosyntheses through the addition
of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also
capable of adding other monosaccharides to the spinosyn aglycone (AGL)
from TDP-sugars; however, the substitution of UDP-d-glucose
for TDP-d-glucose as the donor substrate is known to result
in a >60000-fold reduction in <i>k</i><sub>cat</sub>.
Here,
we report the structure of SpnG at 1.65 Ã… resolution, SpnG bound
to TDP at 1.86 Ã… resolution, and SpnG bound to AGL at 1.70 Ã…
resolution. The SpnG–TDP complex reveals how SpnG employs N202
to discriminate between TDP- and UDP-sugars. A conformational change
of several residues in the active site is promoted by the binding
of TDP. The SpnG–AGL complex shows that the binding of AGL
is mediated via hydrophobic interactions and that H13, the potential
catalytic base, is within 3 Ã… of the nucleophilic 9-OH group
of AGL. A model for the Michaelis complex was constructed to reveal
the features that allow SpnG to transfer diverse sugars; it also revealed
that the rhamnosyl moiety is in a skew-boat conformation during the
transfer reaction
A Double-Hotdog with a New Trick: Structure and Mechanism of the <i>trans</i>-Acyltransferase Polyketide Synthase Enoyl-isomerase
Many
polyketide natural products exhibit invaluable medicinal properties,
yet much remains to be understood regarding the machinery responsible
for their biosynthesis. The recently discovered <i>trans</i>-acyltransferase polyketide synthases employ processing enzymes that
catalyze modifications unique from those of the classical <i>cis</i>-acyltransferase polyketide synthases. The enoyl-isomerase
domains of these megasynthases shift double bonds and are well-represented
by an enzyme that helps forge the triene system within the antibiotic
produced by the prototypical bacillaene synthase. This first crystal
structure of an enoyl-isomerase, at 1.73 Ã… resolution, not only
revealed relationships between this class of enzymes and dehydratases
but also guided an investigation into the mechanism of double bond
migration. The catalytic histidine, positioned differently from that
of dehydratases, was demonstrated to independently shuttle a proton
between the γ- and α-positions of the intermediate. This
unprecedented mechanism highlights the catalytic diversity of divergent
enzymes within <i>trans-</i>acyltransferase polyketide synthases
Molecular Dynamics Studies of Modular Polyketide Synthase Ketoreductase Stereospecificity
Ketoreductases (KRs) from modular
polyketide synthases (PKSs) can
perform stereospecific catalysis, selecting a polyketide with a d- or l-α-methyl substituent for NADPH-mediated
reduction. In this report, molecular dynamics (MD) simulations were
performed to investigate the interactions that control stereospecificity.
We studied the A1-type KR from the second module of the amphotericin
PKS (A1), which is known to be stereospecific for a d-α-methyl-substituted
diketide substrate (dkD). MD simulations of two ternary complexes
comprised of the enzyme, NADPH, and either the correct substrate,
dkD, or its enantiomer (dkL) were performed. The coordinates for the
A1/NADPH binary complex were obtained from a crystal structure (PDB
entry 3MJS), and substrates were modeled in the binding pocket in
conformations appropriate for reduction. Simulations were intended
to reproduce the initial weak binding of the polyketide substrate
to the enzyme. Long (tens of nanoseconds) MD simulations show that
the correct substrate is retained in a conformation closer to the
reactive configuration. Many short (up to a nanosecond) MD runs starting
from the initial structures display evidence that Q364, three residues
N-terminal to the catalytic tyrosine, forms a hydrogen bond to the
incorrect dkL substrate to yield an unreactive conformation that is
more favorable than the reactive configuration. This interaction is
not as strong for dkD, as the d-α-methyl substituent
is positioned between the glutamine and the reactive site. This result
correlates with experimental findings [Zheng, J., et al. (2010) <i>Structure 18</i>, 913–922] in which a Q364H mutant was
observed to lose stereospecificity