Integrative structural and functional analysis of human malic enzyme 3: A potential therapeutic target for pancreatic cancer.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Crystal structure of AdoMet radical enzyme 7-carboxy-7-deazaguanine synthase from Escherichia coli suggests how modifications near [4Fe-4S] cluster engender flavodoxin specificity.

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 CX3 CXϕC motif, to bind a [4Fe-4S] cluster, and a partial (ß/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. Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7-carboxy-7-deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.

Structural and spectroscopic analyses of the sporulation killing factor biosynthetic enzyme SkfB, a bacterial AdoMet radical sactisynthase.

Sactipeptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain a unique thioether bond, referred to as a sactionine linkage, between the sulfur atom of a cysteine residue and the α-carbon of an acceptor residue. These linkages are formed via radical chemistry and are essential for the spermicidal, antifungal, and antibacterial properties of sactipeptides. Enzymes that form these linkages, called sactisynthases, are AdoMet radical enzymes in the SPASM/Twitch subgroup whose structures are incompletely characterized. Here, we present the X-ray crystal structure to 1.29-Å resolution and Mössbauer analysis of SkfB, a sactisynthase from Bacillus subtilis involved in making sporulation killing factor (SKF). We found that SkfB is a modular enzyme with an N-terminal peptide-binding domain comprising a RiPP recognition element (RRE), a middle domain that forms a classic AdoMet radical partial (ß/α)6 barrel structure and displays AdoMet bound to the [4Fe-4S] cluster, and a C-terminal region characteristic of the so-called Twitch domain housing an auxiliary iron-sulfur cluster. Notably, both crystallography and Mössbauer analyses suggest that SkfB can bind a [2Fe-2S] cluster at the auxiliary cluster site, which has been observed only once before in a SPASM/Twitch auxiliary cluster site in the structure of another AdoMet radical enzyme, the pyrroloquinoline quinone biosynthesis enzyme PqqE. Taken together, our findings indicate that SkfB from B. subtilis represents a unique enzyme containing several structural features observed in other AdoMet radical enzymes.

A Rich Man, Poor Man Story of S-Adenosylmethionine and Cobalamin Revisited.

S-adenosylmethionine (AdoMet) has been referred to as both "a poor man's adenosylcobalamin (AdoCbl)" and "a rich man's AdoCbl," but today, with the ever-increasing number of functions attributed to each cofactor, both appear equally rich and surprising. The recent characterization of an organometallic species in an AdoMet radical enzyme suggests that the line that differentiates them in nature will be constantly challenged. Here, we compare and contrast AdoMet and cobalamin (Cbl) and consider why Cbl-dependent AdoMet radical enzymes require two cofactors that are so similar in their reactivity. We further carry out structural comparisons employing the recently determined crystal structure of oxetanocin-A biosynthetic enzyme OxsB, the first three-dimensional structural data on a Cbl-dependent AdoMet radical enzyme. We find that the structural motifs responsible for housing the AdoMet radical machinery are largely conserved, whereas the motifs responsible for binding additional cofactors are much more varied.

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 13C 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.

7-Carboxy-7-deazaguanine Synthase: A Radical S-Adenosyl-l-methionine Enzyme with Polar Tendencies.

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.

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.

Microwave-accelerated surface modification of plasmonic gold thin films with self-assembled monolayers of alkanethiols.

A rapid surface modification technique for the formation of self-assembled monolayers (SAMs) of alkanethiols on gold thin films using microwave heating in <10 min is reported. In this regard, SAMs of two model alkanethiols, 11-mercaptoundecanoic acid (11-MUDA, to generate a hydrophilic surface) and undecanethiol (UDET, a hydrophobic surface), were successfully formed on gold thin films using selective microwave heating in (1) a semicontinuous fashion and (2) a continuous fashion at room temperature (24 h, control experiment, no microwave heating). The formation of SAMs of 11-MUDA and UDET was confirmed by contact angle measurements, Fourier transform infrared (FT-IR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The contact angles for water on SAMs formed by the selective microwave heating and conventional room temperature incubation technique (24 h) were measured to be similar for 11-MUDA and UDET. FT-IR spectroscopy results confirmed that the internal structures of SAMs prepared using both microwave heating and room temperature were similar. XPS results revealed that the organic and sulfate contaminants found on bare gold thin films were replaced by SAMs after the surface modification process had been conducted using both microwave heating and room temperature.

Rapid crystallization of L-arginine acetate on engineered surfaces using metal-assisted and microwave-accelerated evaporative crystallization().

We report the application of our newly described crystallization technique, which employs silver island films (SIFs) and microwave heating, to rapid crystallization of L-arginine acetate (LAA). Using our technique, LAA crystals (~ 1.2 mm in length) were grown from a 20 µl solution in 1 min on surface functionalized SIFs. In control experiments (glass slides and at room temperature) the growth of LAA crystals (0.1-0.3 mm) took ~ 55 min.

Rapid crystallization of glycine using metal-assisted and microwave-accelerated evaporative crystallization: the effect of engineered surfaces and sample volume.

Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC), is a new approach to crystallization of drug compounds, amino acids, DNA and proteins. In this work, we report our additional findings on the effect of engineered surfaces and sample volume on the rapid crystallization of glycine. With the use of hydrophilic functionalized surfaces and the MA-MAEC technique, glycine crystals ~1 mm in size were grown in 35 seconds with 100% selectivity for the α-form.The use of moderately hydrophobic surfaces resulted in the growth of glycine crystals only at room temperature. An increase in volume of initial glycine solution (5-100 µL) resulted in an increase in crystal size without a significant increase in total crystallization time. Raman spectroscopy and powder X-ray diffraction results demonstrated that the glycine crystals grown on engineered surfaces were structurally identical to those grown using conventional evaporative crystallization.

Rapid and sensitive detection of troponin I in human whole blood samples by using silver nanoparticle films and microwave heating.

BACKGROUND: Cardiovascular diseases are among the leading causes of mortality in developed countries. It is widely recognized that troponin I (TnI) can be used for the assessment of a myocardial infarction. METHODS: We investigated the use of the microwave-accelerated and metal-enhanced fluorescence (MA-MEF), a technique based on the combined use of low-power microwave heating, silver nanoparticle films (SNFs), and fluorescence spectroscopy for the detection of TnI from human whole blood samples. SNFs were deposited onto amine-modified glass microscope slides by use of Tollen's reaction scheme and characterized by optical absorption spectroscopy and scanning electron microscopy. The detection of TnI from buffer solutions and human whole blood samples on SNFs was carried out by using fluorescence-based immunoassays at room temperature (control immunoassay, 2 h total assay time) or microwave heating (MA-MEF-based immunoassay, 1 min total assay time). RESULTS: We found that the lower limits of detection for TnI from buffer solutions in the control immunoassay and MA-MEF-based immunoassay were 0.1 µg/L and 0.005 µg/L, respectively. However, we were unable to detect TnI in whole blood samples in the control immunoassays owing to the coagulation of whole blood within 5 min of the incubation step. The use of the MA-MEF technique allowed detection of TnI from whole blood samples in 1 min with a lower detection limit of 0.05 µg/L. CONCLUSIONS: The MA-MEF-based immunoassay is one of the fastest reported quantitative detection methodos for detection of TnI in human whole blood and has low detection limits similar to those obtained with commercially available immunoassays.

Quantitative Comparison of Protein Surface Coverage on Glass Slides and Silver Island Films in Metal-Enhanced Fluorescence-based Biosensing Applications.

The use of Metal-Enhanced Fluorescence (MEF) phenomenon in fluorescence-based bioassays affords for increased sensitivity to be realized by incorporating metal nanoparticles onto planar surfaces. The close-range interactions of metal-fluorophores result in increased fluorescence emission from the bioassays, which in turn affords for the detection of target biomolecules at lower concentrations. Moreover, the use of silver nanoparticles increases the photostability of fluorophores improving the detectability of fluorescence emission under prolonged use of excitation light. Although numerous reports on MEF-based biosensing applications exist, the contribution of protein coverage on Silver Island Films (SIFs) on the increased fluorescence emission was never investigated. This work presents our findings on the quantitative comparison of protein surface coverage on SIFs and blank glass slides. In this regard, identical protein bioassay for a model protein (biotinylated bovine serum albumin, b-BSA) on these surfaces is constructed and the relative extent of protein surface coverage on SIFs and blank glass slides was determined using radio-labeled biomolecules. It was found that the total scintillation counts on SIFs and blank glass slides were similar for BSA concentrations ranging from 1 µM to 1 pM, which implies that increased fluorescence in MEF-based biosensing applications is only due to metal-fluorophore interactions.