Metalloenzymes, Enzymes, & Medicine
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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 ofChristopher 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 controlled.
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.