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Metalloenzymes, Enzymes, &
Medicine |
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Deoxynucleotide
Biosynthesis |
Ribonucleotide
reductases (RNRs) catalyze an essential step in DNA biosynthesis,
the conversion of ribonucleotides to deoxyribonucleotides.
RNR inhibition reduces cellular pools of deoxynucleoside triphosphates
(dNTPs), consequently impairing DNA biosynthesis and DNA repair.
This crucial function has stimulated interest in these enzymes
as antitumor, antiviral, and antibacterial drug targets. The Drennan lab has
studied a wide variety of RNR enzymes, which can differ enormously in structure,
oligomeric state, and regulation. Despite their diversity, RNRs all utilize
a conserved chemical mechanism that is initiated by a protein-bound radical.
RNRs are classified by how they generate and store this radical:
Class I RNRs, found in eukaryotes, utilize
an iron and/or manganese cofactor and a stable tyrosyl radical;
class II RNRs, found in bacteria, algae, and archaea, utilize
coenzyme B12 (AdoCbl, adenosylcobalamin); class III RNRs,
found in anaerobic bacteria, utilize an Fe4S4 cluster and
S-adenosylmethionine to generate a glycyl radical. We are
using crystallography to study all three classes of RNRs with
the goal of understanding the molecular basis for substrate
and inhibitor binding, and the allosteric regulation of enzyme
activity.
Recent work in the lab has elucidated the mechanism of allosteric
inhibition in the class Ia RNR from E. coli. This enzyme is the
prototype for understanding the complex chemistry which all these enzymes
use to generate deoxyribonucleotides. By using a variety of techniques in
collaboration with the laboratories of
Francisco Asturias at Scripps and
JoAnne Stubbe at MIT, we have
shown that dATP, a product of the RNR reaction, causes a change in the oligomeric
state of RNR which locks the protein in a conformation in which it cannot perform
the reaction.
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Oligomeric changes in class
Ia RNR regulate activity
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Publications
Ando N, Brignole EJ, Zimanyi CM, Funk MA, Yokoyama K, Asturias FJ, Stubbe J,
Drennan CL. (2011) Structural interconversions modulate activity of Escherichia coli
ribonucleotide reductase. Proc. Natl. Acad. Sci. U.S.A. 108,
21046-21051.
Sintchak, M.D., Arjara, G., Kellogg, B.A., Stubbe,
J., and Drennan, C.L. (2002)
The Crystal Structure of Class II Ribonucleotide Reductase
Reveals How an Allosterically Regulated Monomer Mimics a Dimer,
Nature Structural Biology. 9:293–300. |
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DNA Repair |
The Drennan lab is interested in understanding several aspects of DNA repair and
has combined structural techniques with biochemistry to understand how DNA repair enzymes
function. In collaboration with the lab of Leona Samson at MIT, we have solved
structures of a human DNA repair protein, alkyladenine DNA glycosylase (AAG).
To efficiently repair DNA, 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. We have solved a crystal
structure of this human DNA repair protein that allows us to investigate, for the first
time, a lower-affinity depiction of this enzyme. By combining this new insight with
existing biochemical and structural data, we are able to consider the big picture question
of how DNA binding proteins find their binding sites in the vast expanse of the genome.
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The mechanism of DNA searching and lesion recognition by AAG |
The process known as "adaptive response" allows E. coli to survive
stress induced by a class of highly mutagenic compounds called DNA alkylating
agents. Four proteins are upregulated during the adaptive response, including
the flavin-binding protein AidB, the function of which is still largely
unknown. We have worked with the laboratory of Sean Elliott at BU to apply a wide spectrum
of techniques—including fluorescence anisotropy, analytical ultracentrifugation, and X-ray
crystallography—to show that AidB undergoes a
flavin-dependent transition in oligomerization state from a dimer to a tetramer. These results provide
strong evidence that flavin plays a structural role in the formation of an AidB
tetramer, with potential functional implications.
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AidB flavin-dependent oligomerization
seen by ultracentrifugation |
Publications
Setser JW, Lingaraju GM, Davis CA, Samson LD, Drennan CL. (2012) Searching for DNA
lesions: structural evidence for lower- and higher-affinity DNA binding conformations
of human alkyladenine DNA glycosylase. Biochemistry 51, 382-390.
Full text at ACS Publications
Hamill MJ, Jost M, Wong C, Elliott SJ, Drennan CL. (2011) Flavin-induced oligomerization
in Escherichia coli adaptive response protein AidB. Biochemistry 50,
10159-10169.
Full text at ACS Publications
Lingaraju GM, Davis CA, Setser JW, Samson LD, Drennan CL. (2011) Structural basis
for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4
-ethenocytosine-containing DNA. J. Biol. Chem. 286, 13205-13213.
Full text at JBC.org
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Vitamin
Biosynthesis |
S-adenosylmethionine
(AdoMet) radical enzymes use a 4Fe-4S cluster to generate
an active radical species by reductive cleavage of AdoMet.
This radical is an extraordinary oxidant that can remove a hydrogen atom
from an unactivated substrate, often initiating a cascade of radical-mediated rearrangements.
Substrates of this superfamily of enzymes range from small molecules to entire
proteins to DNA or RNA molecules. The Drennan lab is currently studying several AdoMet radical
enzymes, including those that create the vitamins biotin and lipoate. |
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The Challenging
reaction catalyzed by biotin synthase (BioB) |
Publications
Berkovitch, F., Nicolet, Y., Wan, J.T., Jarrett,
J.T., and Drennan, C.L. (2004) The
Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent
Radical Enzyme, Science. 303:76-79.
Nicolet,
Y., and Drennan, C.L. (2004)
AdoMet Radical Proteins – from Structure to
Evolution – Alignment of Divergent Protein Sequences
Reveals Strong Secondary Structure Element Conservation,
Nucleic Acids Research. 32:4015-4025.
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Natural
Product Biosynthesis |
Non-heme
iron enzymes harness oxygen to perform a wide variety of challenging
chemistry, including hydroxylation, epoxidation, and halogenation.
These enzymes are often employed to perform reactions
in natural product biosynthesis. For example, hydroxypropylphosphonic
acid epoxidase (HppE) performs a key epoxidation reaction
in the biosynthesis of the antibiotic fosfomycin. In collaboration with the lab of
Ben Liu, we have crystallized
the enzyme bound to several unnatural substrates. Using this structural information,
we have revealed a clear picture of the mechanism and the structural basis for
substrate specificity.
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HppE-catalyzed
formation of fosfomycin |
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Substrates bound to HppE
reveal substrate specificity |
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A large number of natural products are synthesized from familiar starting
materials, for example, amino acids. These initial building blocks are often
modified to alter their reactivity or allow them to bind to their targets.
The Drennan lab is interested in the "tailoring" enzymes which generate these
modified amino acids, most of which are important for the activity of the natural
products they are a part of. The non-heme iron enzyme SyrB2 catalyzes a halogenation
reaction that is essential for the activity of syringomycin, an anti-fungal
agent. We are interested in understanding how the structure of enzymes like SyrB2 tunes their
function and specificity.
The halogenation
reaction catalyzed by SyrB2
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Publications
Yun D, Dey M, Higgins LJ, Yan F, Liu H-W, Drennan CL. (2011)
Structural Basis of Regiospecificity of a Mononuclear Iron Enzyme in Antibiotic Fosfomycin Biosynthesis.
J. Am. Chem. Soc. 131, 11262-11269.
Blasiak LC, Vaillancourt FH, Walsh CT,
and Drennan CL. (2006) Crystal
structure of the non-haem iron halogenase SyrB2 in syringomycin
biosynthesis, Nature. 440:368-71.
Higgins
LJ, Yan F, Liu P, Liu HW, and Drennan CL. (2005)
Structural
insight into antibiotic fosfomycin biosynthesis by a mononuclear
iron enzyme, Nature. 437L838-44.
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Flavoenzymes contain an FAD or FMN cofactor and are capable of performing some of the same
complex chemical reactions catalyzed by mononuclear iron and heme enzymes. The
Drennan lab is interested in several flavoenzymes involved in the biosynthesis of
indolocarbazole natural products, including rebeccamycin and staurosporine. The
scaffold of these complex molecules is constructed by the enzyme RebC through the oxidation of two
tryptophans. The FAD-dependent halogenase RebH performs a chlorination of tryptophan
during rebeccamycin production. This reaction is especially interesting in comparison with
the halogenation catalyzed by the non-heme iron enzyme SyrB2
as described above. The Drennan lab is working closely with the lab of
Christopher Walsh
at Harvard Medical School to understand the mechanism of these enzymes: how they
are similar, how the enzymes control the reactive intermediates, and how the
substrate specificity is controled. |
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Chemotherapeutic
rebeccamycin and the halogenase RebH |
Publications
Ryan KS, Chakraborty S, Howard-Jones AR, Walsh CT, Ballou DP, Drennan CL. (2008)
The FAD Cofactor
of RebC Shifts to an IN Conformation upon Flavin Reduction.
Biochemistry. 47:14506-13513.
Ryan KS, Howard-Jones AR, Hamill MJ, Elliott SJ, Walsh CT, Drennan CL. (2007)
Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC.
Proc. Natl. Acad. Sci. U.S.A. 104(39):15311-15316.
Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT.
(2007)
Chlorination by a long-lived intermediate in the mechanism
of flavin-dependent halogenases, Biochemistry.
46:1284-92.
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