Structural insight into the assembly of iron-sulfur clusters and their function in radical generation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.Includes bibliographical references.This thesis addresses two emerging areas in the study of iron-sulfur cluster biochemistry: bioassembly of iron-sulfur clusters, and their involvement in initiation of radical chemistry. The structure of a cysteine desulfurase involved in cluster bioassembly in the cyanobacterium Synechocystis PCC sp. 6803 was solved by X-ray crystallography and analyzed in terms of its mechanistic implications. We found that the active site cysteine responsible for the direct removal of sulfur from substrate cysteine is located on a short, well-ordered loop, consistent with structures solved of homologous proteins. The length of this loop is thought to restrain the active site cysteine, interfering with its ability to affect catalysis. Our results are consistent with the theory that this cysteine desulfurase requires an accessory protein for fully activity in vivo. Two structures of pyruvate formate-lyase activating enzyme from Escherichia coli, an Sadenosylmethionine radical enzyme, were also solved by X-ray crystallography, providing the first structure of an activase from this family of enzymes. These structures revealed the enzyme's active site and the residues involved in binding and orienting substrate for hydrogen atom abstraction. Comparison of the structures of the substrate-free and substrate-bound forms of the enzyme identified a conformational change associated with substrate binding. Detailed analyses of the structure of pyruvate formatelyase activating enzyme were carried out to provide insight into catalysis. These structures were also analyzed in comparison with other S-adenosylmethionine radical enzyme structures to more clearly understand the structural basis for reactivity in this superfamily.by Jessica L. Vey.Ph.D

Structural diversity in the AdoMet radical enzyme superfamily

AdoMet radical enzymes are involved in processes such as cofactor biosynthesis, anaerobic metabolism, and natural product biosynthesis. These enzymes utilize the reductive cleavage of S-adenosylmethionine (AdoMet) to afford l-methionine and a transient 5′-deoxyadenosyl radical, which subsequently generates a substrate radical species. By harnessing radical reactivity, the AdoMet radical enzyme superfamily is responsible for an incredible diversity of chemical transformations. Structural analysis reveals that family members adopt a full or partial Triose-phosphate Isomerase Mutase (TIM) barrel protein fold, containing core motifs responsible for binding a catalytic [4Fe–4S] cluster and AdoMet. Here we evaluate over twenty structures of AdoMet radical enzymes and classify them into two categories: ‘traditional’ and ‘ThiC-like’ (named for the structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (ThiC)). In light of new structural data, we reexamine the ‘traditional’ structural motifs responsible for binding the [4Fe–4S] cluster and AdoMet, and compare and contrast these motifs with the ThiC case. We also review how structural data combine with biochemical, spectroscopic, and computational data to help us understand key features of this enzyme superfamily, such as the energetics, the triggering, and the molecular mechanisms of AdoMet reductive cleavage. This article is part of a Special Issue entitled: Radical SAM Enzymes and Radical Enzymology.Wellcome Trust (London, England) (091162/Z/10/Z)National Science Foundation (U.S.) (NSF Grant MCB-0543833)Howard Hughes Medical Institute (Investigator

2017 Research & Innovation Day Program

A one day showcase of applied research, social innovation, scholarship projects and activities.https://first.fanshawec.ca/cri_cripublications/1004/thumbnail.jp

Structural Insights into Radical Generation by the Radical SAM Superfamily

Table of Contents Table of Contents 1. Introduction 2. Unresolved Questions in the Radical SAM Enzyme Field 3. Highlighted Radical SAM Enzymes 3.1. Pyruvate Formate-Lyase Activating Enzyme (PFL-AE) 3.2. Oxygen-Independent Coproporphyrinogen III Oxidase (HemN) 3.3. Biotin Synthase (BioB) 3.4. Molybdenum Cofactor Biosynthesis Protein MoaA 3.5. Lysine Aminomutase (LAM) 3.6. Wye-Base Biosynthetic Protein TYW1 3.7. [Fe−Fe] Hydrogenase Maturase Protein HydE 4. Overall Fold 4.1. Radical SAM Core 4.2. Protein Elements Outside of the Radical SAM Core 5. FeS cluster 5.1. Location of the 4Fe−4S Cluster Binding Site 5.2. Environment Surrounding the Cluster 5.3. Interactions between the 4Fe−4S Cluster and AdoMet 6. AdoMet Binding 6.1. AdoMet Conformation 6.2. General Properties of the AdoMet Binding Site 6.3. Overall Description of the AdoMet Binding Site 6.3.1. AdoMet Methionyl Moiety 6.3.2. AdoMet Ribose 6.3.3. AdoMet Adenine Moiety 6.4. AdoMet Binding Motifs in the Radical SAM Superfamily 6.5. Deviations and Variations in AdoMet Binding between the Subfamilies 7. Implications of AdoMet Binding Site Architecture on Function and Reactivity 7.1. Tailoring the Reaction to Specific Substrates 7.2. AdoMet Usage as Cofactor or Cosubstrate 7.3. AdoMet Reaction Stoichiometry 7.4. HemN’s Second AdoMet (SAM2) 8. Substrate Binding to Radical SAM Enzymes 8.1. Positioning of the Substrate 8.2. Additional Cofactors in Some Radical SAM Substrate Binding Sites 8.3. Conformational Changes Associated with Substrate Binding in Radical SAM Enzymes 9. Reductant Binding in Radical SAM Enzymes 10. Other Known AdoMet-Binding Protein Folds 11. ConclusionsMassachusetts Institute of Technology (William Asbjornsen Albert Memorial Fellowship)Howard Hughes Medical Institute (Investigator

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B₁₂ enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na⁺ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by ²³Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M⁺ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[¹³C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li⁺ to Cs⁺, PFL-AE activity sharply maximizes at K⁺, with NH₄⁺ closely matching the efficacy of K⁺. PFL-AE is thus a type I M⁺-activated enzyme whose M⁺ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster