Radical-mediated ring contraction in the biosynthesis of 7-deazapurines

Pyrrolopyrimidine containing natural products are widely distributed in Nature. The biosynthesis of the 7-deazapurine moiety that is common to all pyrrolopyrimidines entails multiple steps, one of which is a complex radical-mediated ring contraction reaction catalyzed by CDG synthase. Herein we review the biosynthetic pathways of deazapurines, focusing on the biochemical and structural insights into CDG synthase

A research-inspired laboratory sequence investigating acquired drug resistance

Here, we present a six-session laboratory exercise designed to introduce students to standard biochemical techniques in the context of investigating a high impact research topic, acquired resistance to the cancer drug Gleevec. Students express a Gleevec-resistant mutant of the Abelson tyrosine kinase domain, the active domain of an oncogenic protein implicated in chronic myelogenous leukemia, and investigate the kinase activity of wild type and mutant enzyme in the presence of two cancer drugs. Techniques covered include protein expression, purification, and gel analysis, kinase activity assays, and protein structure viewing. The exercises provide students with a hands-on understanding of the impact of biochemistry on human health, and demonstrate their potential as the next generation of investigators.Howard Hughes Medical Institut

A structural phylogeny for understanding 2-oxoacid oxidoreductase function

2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (~-500 mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxidoreductase, structure-function relationships in this superfamily have been expanded and re-evaluated. Additionally, structural motifs have been defined that better serve to distinguish one OFOR subfamily from another and potentially uncover novel OFORs.National Institutes of Health (U.S.) (Grant GM069857)National Science Foundation (U.S.) (Grant 1122374

SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N- and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily.Massachusetts Institute of Technology. Office of the Dean for Graduate Education (Fellowship)National Science Foundation (U.S.) (Grant MCB-0543833

Metalloprotein Crystallography: More than a Structure

Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25–50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV–vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.National Institutes of Health (U.S.) (Grant GM069857)National Institutes of Health (U.S.) (Grant F32-GM099257)National Institutes of Health (U.S.) (Grant F32-GM108189

Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from

Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases

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

Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine to form the posttranscriptional modification, 4-demethylwyosine, in situ on transfer RNA (tRNA). Two mechanisms have been proposed for this transformation, with one of the possible mechanisms invoking a Schiff base intermediate formed between a conserved lysine residue and pyruvate. Utilizing a combination of mass spectrometry and X-ray crystallography, we have obtained evidence to support the formation of a Schiff base lysine adduct in TYW1. When 13 C labeled pyruvate is used, the mass shift of the adduct matches that of the labeled pyruvate, indicating that pyruvate is the source of the adduct. Furthermore, a crystal structure of TYW1 provides visualization of the Schiff base lysine-pyruvate adduct, which is positioned directly adjacent to the auxiliary [4Fe-4S] cluster. The adduct coordinates the unique iron of the auxiliary cluster through the lysine nitrogen and a carboxylate oxygen, reminiscent of how the radical SAM [4Fe-4S] cluster is coordinated by SAM. The structure provides insight into the binding site for tRNA and further suggests how radical SAM chemistry can be combined with Schiff base chemistry for RNA modification.National Science Foundation (U.S.) (Grant 1122374

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production.National Science Foundation (U.S.) (Grant 0645960

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.United States. National Institutes of Health (F32-GM108189