8 research outputs found
Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent
The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473
Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase
To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.National Institutes of Health (U.S.) (grant no. P30-ES002109)National Institutes of Health (U.S.) (grant no. GM65337)National Institutes of Health (U.S.) (grant no. GM65337-03S2)National Institutes of Health (U.S.) (grant no. CA055042)National Institutes of Health (U.S.) (grant no. CA092584)Repligen Corporation (KIICR Graduate Fellowship
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Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent <i>N</i>‑Hydroxylase
The soil actinomycete <i>Kutzneria</i> sp. 744 produces
a class of highly decorated hexadepsipeptides, which represent a new
chemical scaffold that has both antimicrobial and antifungal properties.
These natural products, known as kutznerides, are created via nonribosomal
peptide synthesis using various derivatized amino acids. The piperazic
acid moiety contained in the kutzneride scaffold, which is vital for
its antibiotic activity, has been shown to derive from the hydroxylated
product of l-ornithine, l-<i>N</i><sup>5</sup>-hydroxyornithine. The production of this hydroxylated species
is catalyzed by the action of an FAD- and NAD(P)H-dependent <i>N</i>-hydroxylase known as KtzI. We have been able to structurally
characterize KtzI in several states along its catalytic trajectory,
and by pairing these snapshots with the biochemical and structural
data already available for this enzyme class, we propose a structurally
based reaction mechanism that includes novel conformational changes
of both the protein backbone and the flavin cofactor. Further, we
were able to recapitulate these conformational changes in the protein
crystal, displaying their chemical competence. Our series of structures,
with corroborating biochemical and spectroscopic data collected by
us and others, affords mechanistic insight into this relatively new
class of flavin-dependent hydroxylases and adds another layer to the
complexity of flavoenzymes
Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase
To efficiently repair DNA, human alkyladenine DNA glycosylase
(AAG)
must search the million-fold excess of unmodified DNA bases to find
a handful of DNA lesions. Such a search can be facilitated by the
ability of glycosylases, like AAG, to interact with DNA using two
affinities: a lower-affinity interaction in a searching process and
a higher-affinity interaction for catalytic repair. Here, we present
crystal structures of AAG trapped in two DNA-bound states. The lower-affinity
depiction allows us to investigate, for the first time, the conformation
of this protein in the absence of a tightly bound DNA adduct. We find
that active site residues of AAG involved in binding lesion bases
are in a disordered state. Furthermore, two loops that contribute
significantly to the positive electrostatic surface of AAG are disordered.
Additionally, a higher-affinity state of AAG captured here provides
a fortuitous snapshot of how this enzyme interacts with a DNA adduct
that resembles a one-base loop
Discovery of Benzotriazolo[4,3‑<i>d</i>][1,4]diazepines as Orally Active Inhibitors of BET Bromodomains
Inhibition of the bromodomains of
the BET family, of which BRD4 is a member, has been shown to decrease
myc and interleukin (IL) 6 <i>in vivo</i>, markers that
are of therapeutic relevance to cancer and inflammatory disease, respectively.
Herein we report substituted benzo[<i>b</i>]isoxazolo[4,5-<i>d</i>]azepines and benzotriazolo[4,3-<i>d</i>][1,4]diazepines
as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds
from these series were potent and selective in cells, and subsequent
optimization of microsomal stability yielded representatives that
demonstrated dose- and time-dependent reduction of plasma IL-6 in
mice
Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637)
CBP and EP300 are highly homologous,
bromodomain-containing transcription
coactivators involved in numerous cellular pathways relevant to oncology.
As part of our effort to explore the potential therapeutic implications
of selectively targeting bromodomains, we set out to identify a CBP/EP300
bromodomain inhibitor that was potent both <i>in vitro</i> and in cellular target engagement assays and was selective over
the other members of the bromodomain family. Reported here is a series
of cell-potent and selective probes of the CBP/EP300 bromodomains,
derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2<i>H</i>-benzo[<i>b</i>][1,4]diazepin-2-one