William E. Broderick

Structural Basis for Glycyl Radical Formation By Pyruvate Formate-Lyase Activating Enzyme

Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G734 of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G734. Our structures provide fundamental insights into the interactions between the activase and the G734 loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G734 of pyruvate formate-lyase.

A Molecular Ferromagnet with a Curie Temperature of 6.2 Kelvin: [Mn(C5(CH3)5)2]+[TCNQ]-.

The study of magnetic phase transitions in insulating molecular solids provides new insights into mechanisms of magnetic coupling in the solid state and into critical phenomena associated with these transitions. Only a few such materials are known to display cooperative magnetic properties. The use of high-spin molecular components would enhance intermolecular spin-spin interactions and thus a series of chargetransfer (CT) salts have been synthesized that utilize the spin S = 1 molecular cation, [Mn(C5(CH3)5)2]+ (decamethylmanganocenium). The structure and cooperative magnetic behavior of [Mn(C5(CH3)5)2]+[TCNQ- (decamethylmanganocenium 7,7,8,8-tetracyano-p-quinodimethanide) are reported. This salt is a bulk molecular ferromagnet with the highest critical (Curie) temperature (Tc = 6.2 K) and coercive field (3.6 x 103 gauss), yet reported for such a material.

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

In an effort to determine the specific protein component(s) responsible for in vitro activation of the [FeFe] hydrogenase (HydA), the individual maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum were purified from heterologous expressions in Escherichia coli. Our results demonstrate that HydF isolated from a strain expressing all three maturation proteins is sufficient to confer hydrogenase activity to purified inactive heterologously expressed HydA (expressed in the absence of HydE, HydF, and HydG). These results represent the first in vitro maturation of [FeFe] hydrogenase with purified proteins, and suggest that HydF functions as a scaffold upon which an H‐cluster intermediate is synthesized.

Spore Photoproduct Lyase Catalyzes Specific Repair of the 5R but not the 5S Spore Photoproduct

Bacterial spores are remarkable in their resistance to chemical and physical stresses, including exposure to UV radiation. The unusual UV resistance of bacterial spores is a result of the unique photochemistry of spore DNA, which results in accumulation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), coupled with the efficient repair of accumulated damage by the enzyme spore photoproduct lyase (SPL). SPL is a member of the radical AdoMet superfamily of enzymes, and utilizes an iron-sulfur cluster and S-adenosylmethionine to repair SP by a direct reversal mechanism initiated by H atom abstraction from C-6 of the thymine dimer. While two distinct diastereomers of SP (5R or 5S) could in principle be formed upon UV irradiation of bacterial spores, only the 5R configuration is possible for SP formed from adjacent thymines in double helical DNA, due to the constraints imposed by the DNA structure; the 5S configuration is possible in less well-defined DNA structures or as an interstrand cross-link. We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemically defined SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. The observation that 5R-SP, but not 5S-SP, is a substrate for SPL is consistent with the expectation that 5R is the SP isomer produced in vivo upon UV irradiation of bacterial spore DNA.

Structural studies of the interaction of S‐adenosylmethionine with the [4Fe‐4S] clusters in biotin synthase and pyruvate formate‐lyase activating enzyme

The diverse reactions catalyzed by the radical‐SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S‐adenosyl‐L‐methionine (SAM) by a reduced [4Fe‐4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe‐4S] clusters in several of the radical‐SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X‐ray absorption spectroscopy (Se‐XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se‐SAM) and an Fe atom of the [4Fe‐4S] cluster of lysine‐2,3‐aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate‐lyase activating enzyme (PFL‐AE) and biotin synthase (BioB), two additional members of the radical‐SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe‐Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well‐defined site close to the cluster in PFL‐AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.

Radical SAM catalysis via an organometallic intermediate with an Fe–[5′-C]-deoxyadenosyl bond

Catching a radical in action Many enzymes catalyze reactions through the production of radical intermediates. Radical SAM enzymes, the largest superfamily of enzymes in nature, do this by using an iron-sulfur cluster to cleave S-adenosylmethionine and produce a radical intermediate. Using freeze quenching, Horitani et al. were able to trap a previously unseen radical intermediate from bacterial pyruvate formate-lyase activating enzyme. Spectroscopy revealed that the intermediate consists of a short-lived covalent bond between the terminal carbon of 5′-deoxyadenosyl and the single iron atom of the iron-sulfur cluster. Not only does the observation of this radical expand our mechanistic understanding of radical SAM enzymes, but it expands the range of enzyme active sites or cofactors that function through an organometallic center. Science, this issue p. 822 Freeze-quench experiments trap a radical intermediate on pyruvate formate-lyase activating enzyme. Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5′-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and 13C, 57Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5′ carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.

Chemoselective deprotection of triethylsilyl ethers.

An efficient and selective method was developed for the deprotection of triethylsilyl (TES) ethers using formic acid in methanol (5–10%) or in methylene chloride 2–5%) with excellent yields. TES ethers are selectively deprotected to the corresponding alcohols in high yields using formic acid in methanol under mild reaction conditions. Other hydroxyl protecting groups like t-butyldimethylsilyl (TBDMS) remain unaffected.

An Efficient Deprotection of N-Trimethylsilylethoxymethyl (SEM) Groups From Dinucleosides and Dinucleotides

A convenient and efficient method for deprotection of N-(trimethyl)silylethoxymethyl (SEM) groups from thymidine dinucleoside and dinucleotide has been achieved. The SEM groups were easily removed in excellent yields from protected nucleosides, dinucleosides, and dinucleotides.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.