Objective To investigate whether placebo effects can experimentally be separated into the response to three components-assessment and observation, a therapeutic ritual (placebo treatment), and a supportive patient-practitioner relationship-and then progressively combined to produce incremental clinical improvement in patients with irritable bowel syndrome. To assess the relative magnitude of these components.

No abstract

Arylsulfatases require a maturating enzyme to perform a co-or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidylsubstrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteinerich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.iron-sulfur cluster fold | radical SAM dehydrogenase

Metabolic balance studies show that germfree and conventional Sprague-Dawley rats synthesize nitrate. Equivalent results for germfree and conventional rats eliminate the microflora as obligatory components of nitrate production. Nitrate synthesis appears to be a mammalian process.

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. Members of the S-adenosylmethionine (SAM)2 radical superfamily catalyze a wide variety of radical-mediated reactions, including complex chemical transformations and rearrangements; modifications of peptides, DNA, and RNA; dehydrogenations; and sulfur insertions (1). Despite this diversity, there are unifying structural and mechanistic themes. For instance, SAM radical enzymes typically bind a [4Fe-4S] cluster using a conserved CX 3 CXC motif (where is an aromatic residue). This motif provides three cysteine ligands to the iron atoms of the cluster, with the fourth ligand coming from the bidentate coordination of SAM to the unique iron (2, 3). Direct ligation of SAM to the cluster facilitates reductive cleavage of the C-S bond through an inner sphere electron transfer event, forming methionine and a 5Ј-deoxyadenosyl radical (5Ј-dAdo ⅐ ) (Fig. 1A) (4). The abstraction of a hydrogen atom from the substrate by 5Ј-dAdo ⅐ , producing a substrate radical, ends the mechanistic similarity between enzymes of this superfamily; each enzyme utilizes a different mechanism to generate product. Structures of the first seven members of the SAM radical superfamily were used to define a core fold for binding SAM and for the generation of 5Ј-dAdo ⅐ species. This core consists of a partial (␤/␣) 6 triose-phosphate isomerase (TIM) barrel (5). Outside of the core fold, the structure can vary greatly, with Nand C-terminal extensions that are functionalized for binding other cofactors or substrates. The SPASM subfamily is an example of a functionalized C-terminal extension for the binding of two auxiliary clusters.Haft and Basu (6, 7) recognized that enzymes with this C-terminal extension appear to be involved in the modification of ribosomally translated peptides. This subclass is referred to as SPASM after the biochemically characterized members, AlbA, PqqE, anSMEs, and MftC, which are involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively. The SPASM subfamily, accession TIGR04085, is composed of 281 sequences. However, recent similarity network analysis by ...

The extraction, isolation and characterization of 29 natural products contained in Ginkgo biloba have been described, which we have now tested for their in-vitro capacity to inhibit the five major human cytochrome P450 (CYP) isoforms in human liver microsomes. Weak or negligible inhibitory activity was found for the terpene trilactones (ginkgolides A, B, C and J, and bilobalide), and the flavonol glycosides. However 50% inhibitory activity (IC50) was found at concentrations less than 10 microg L(-1) for the flavonol aglycones (kaempferol, quercetin, apigenin, myricetin, tamarixetin) with CYP1A2 and CYP3A. Quercetin, the biflavone amentoflavone, sesamin, as well as (Z,Z)-4,4'-(1,4-pentadiene-1,5-diyl)diphenol and 3-nonadec-8-enyl-benzene-1,2-diol, were also inhibitors of CYP2C9. The IC50 of amentoflavone for CYP2C9 was 0.019 microg mL(-1) (0.035 microM). Thus, the principal components of Ginkgo biloba preparations in clinical use (terpene trilactones and flavonol glycosides) do not significantly inhibit these human CYPs in-vitro. However, flavonol aglycones, the biflavonol amentoflavone and several other non-glycosidic constituents are significant in-vitro inhibitors of CYP. The clinical importance of these potential inhibitors will depend on their amounts in ginkgo preparations sold to the public, and the extent to which their bioavailability allows them to reach the CYP enzymes in-situ.

Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chemistry; the remainder of the reaction mechanism, especially the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resolution crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-containing 8-mer peptide substrate and 1 eq AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissociates from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.iron-sulfur cluster | radical SAM enzyme | lipoic acid T he functionalization of aliphatic carbon centers is widely regarded as one of the most kinetically challenging reactions in nature, a striking example of which is found in the biosynthesis of the lipoyl cofactor, famous for its central role as the "swinging arm" of the pyruvate dehydrogenase enzyme complex. Lipoyl synthase (LipA) generates the lipoyl cofactor by insertion of two sulfur atoms at C6 and C8 of a protein-bound n-octanoyl chain, sites distal from the nearest functionality (1-4). LipA and the closely related biotin synthase (BioB) (Scheme 1) are founding members of the ever-expanding S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily that uses a [4Fe-4S] cluster to reductively cleave the C5′-S bond of AdoMet, generating methionine and a 5′-deoxyadenosyl radical (5′-dA•), a powerful oxidant (5, 6). LipA requires 2 eq AdoMet-one per sulfur insertion-and two sulfur atoms to produce 1 eq lipoyl product through radical-based chemistry (4). One of the most controversial aspects of the LipA and BioB reactions is the source of the sulfur. AdoMet radical enzymes that catalyze sulfur insertion always seem to have an additional iron-sulfur (Fe/S) cluster: a [2Fe-2S] cluster in BioB (7), a [4Fe-4S] cluster in LipA (8), and a [4Fe-4S] cluster in the methylthiotransferases RimO and MiaB (9, 10). These auxiliary clusters in LipA and BioB have been proposed to be cannibalized during turnover to supply the inserted sulfur atom(s) (8, 11-13), a proposal that has not enjoyed univ...

The transformation of digitalis from a folk medicine, foxglove, to a modern drug, digoxin, illustrates principles of modern pharmacology that have helped make drugs safer and more effective. Digitalis was improved because its preparation was standardized, first by bioassay and then by chemical methods; however, few of today's herbs are standardized by methods that can ensure a consistent product and, hence, consistent safety and efficacy profiles. Many herbs have been evaluated in randomized, controlled trials, and several-St. John's wort and ginkgo, for example-are apparently effective. Yet, many trials of herbs have limited value because of poor design, small samples, and, above all, use of products of uncertain composition and consistency. The uncertain composition of many herbal products raises questions about their safety, as does evidence indicating that herbs may have harmful interactions with prescription drugs. Such adverse effects of herbs are probably underreported. Meanwhile, systematic studies, such as those identifying adverse reactions to drugs, are hindered because herbal preparations are not standardized-one brand of St. John's wort, for example, will differ chemically from anotherand, unlike for prescription drugs, there are no databases linking herb consumption to later medical problems. Since herbal medicines are regulated as dietary supplements, they are not subject to the premarketing regulatory clearance required for drugs. The burden of proof is on the U.S. Food and Drug Administration to show a dietary supplement is unsafe, unlike for drugs, which cannot be approved until the manufacturer has demonstrated safety and effectiveness.