Daniel P. Dowling
Massachusetts Institute of Technology
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Featured researches published by Daniel P. Dowling.
Nature Chemical Biology | 2014
Daniel P. Dowling; Nathan A. Bruender; Anthony P. Young; Reid M. McCarty; Vahe Bandarian; Catherine L. Drennan
7-Carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg2+-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5′-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the first crystal structure of a QueE, along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg2+, which directly coordinates to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified (β6/α3) protein core and an expanded cluster-binding motif CX14CX2C.
Annual review of biophysics | 2012
Daniel P. Dowling; Anna K. Croft; Catherine L. Drennan
The ability of enzymes to harness free-radical chemistry allows for some of the most amazing transformations in nature, including reduction of ribonucleotides and carbon skeleton rearrangements. Enzyme cofactors involved in this chemistry can be large and complex, such as adenosylcobalamin (coenzyme B(12)), simpler, such as S-adenosylmethionine and an iron-sulfur cluster (i.e., poor mans B(12)), or very small, such as one nonheme iron atom coordinated by protein ligands. Although the chemistry catalyzed by these enzyme-bound cofactors is unparalleled, it does come at a price. The enzyme must be able to control these radical reactions, preventing unwanted chemistry and protecting the enzyme active site from damage. Here, we consider a set of radical folds: the (β/α)(8) or TIM barrel, combined with a Rossmann domain for coenzyme B(12)-dependent chemistry. Using specific enzyme examples, we consider how nature employs the common TIM barrel fold and its Rossmann domain partner for radical-based chemistry.
Proceedings of the National Academy of Sciences of the United States of America | 2016
Daniel P. Dowling; Yan Kung; Anna K. Croft; Koli Taghizadeh; Wendy L. Kelly; Christopher T. Walsh; Catherine L. Drennan
Significance Here we investigate the structural basis for cyclization activity in hybrid polyketide synthase-nonribosomal peptide synthetases. This first structure of a cyclization (Cy) domain reveals an unexpected location for the enzyme active site, providing a fresh perspective on past mutational studies. Our structures also depict two 20-Å-long channels that create routes for the two tethered substrates to simultaneously reach the buried active site, affording substrate condensation and cyclization. Along with the Cy domain, these structures contain a covalently attached docking domain, providing insight into how protein modules work together to achieve uni-directionality in the biosynthesis of natural products. Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA–F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.
Nucleic Acids Research | 2016
Daniel P. Dowling; Zachary D. Miles; Caroline Köhrer; Stephanie J. Maiocco; Sean J. Elliott; Vahe Bandarian; Catherine L. Drennan
Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis. Our structure of QueG bound to a tRNATyr anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.
Journal of the American Chemical Society | 2017
Nathan A. Bruender; Tsehai A. J. Grell; Daniel P. Dowling; Reid M. McCarty; Catherine L. Drennan; Vahe Bandarian
Radical S-adenosyl-l-methionine (SAM) enzymes are widely distributed and catalyze diverse reactions. SAM binds to the unique iron atom of a site-differentiated [4Fe-4S] cluster and is reductively cleaved to generate a 5′-deoxyadenosyl radical, which initiates turnover. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes a key step in the biosynthesis of 7-deazapurine containing natural products. 6-Carboxypterin (6-CP), an oxidized analogue of the natural substrate 6-carboxy-5,6,7,8-tetrahydropterin (CPH4), is shown to be an alternate substrate for CDG synthase. Under reducing conditions that would promote the reductive cleavage of SAM, 6-CP is turned over to 6-deoxyadenosylpterin (6-dAP), presumably by radical addition of the 5′-deoxyadenosine followed by oxidative decarboxylation to the product. By contrast, in the absence of the strong reductant, dithionite, the carboxylate of 6-CP is esterified to generate 6-carboxypterin-5′-deoxyadenosyl ester (6-CP-dAdo ester). Structural studies with 6-CP and SAM also reveal electron density consistent with the ester product being formed in crystallo. The differential reactivity of 6-CP under reducing and nonreducing conditions highlights the ability of radical SAM enzymes to carry out both polar and radical transformations in the same active site.
Protein Science | 2018
Tsehai A. J. Grell; Benjamin N. Bell; Chi Nguyen; Daniel P. Dowling; Nathan A. Bruender; Vahe Bandarian; Catherine L. Drennan
7‐Carboxy‐7‐deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6‐carboxy‐5,6,7,8‐tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7‐deazaguanine natural products. QueE is a member of the S‐adenosyl‐L‐methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX3CXϕC motif, to bind a [4Fe‐4S] cluster, and a partial (β/α)6 TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster‐binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re‐haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin.
Biochimica et Biophysica Acta | 2012
Daniel P. Dowling; Jessica L. Vey; Anna K. Croft; Catherine L. Drennan
The FASEB Journal | 2015
Daniel P. Dowling; Zachary D. Miles; Caroline Köhrer; Vahe Bandarian; Catherine L. Drennan
The FASEB Journal | 2015
Nathan A. Bruender; Daniel P. Dowling; Catherine L. Drennan; Vahe Bandarian
Prof. Drennan via Erja Kajosalo | 2012
Daniel P. Dowling; Anna K. Croft; Catherine L. Drennan