Crystallographic snapshots of sulfur insertion by lipoyl synthase.

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.

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.

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry.

The 2-deoxy-scyllo-inosamine (DOIA) dehydrogenases are key enzymes in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. In contrast to most DOIA dehydrogenases, which are NAD-dependent, the DOIA dehydrogenase from Bacillus circulans (BtrN) is an S-adenosyl-l-methionine (AdoMet) radical enzyme. To examine how BtrN employs AdoMet radical chemistry, we have determined its structure with AdoMet and substrate to 1.56 Å resolution. We find a previously undescribed modification to the core AdoMet radical fold: instead of the canonical (ß/α)6 architecture, BtrN displays a (ß5/α4) motif. We further find that an auxiliary [4Fe-4S] cluster in BtrN, thought to bind substrate, is instead implicated in substrate-radical oxidation. High structural homology in the auxiliary cluster binding region between BtrN, fellow AdoMet radical dehydrogenase anSME, and molybdenum cofactor biosynthetic enzyme MoaA provides support for the establishment of an AdoMet radical structural motif that is likely common to ~6,400 uncharacterized AdoMet radical enzymes.

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification.

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 peptidyl-substrates, 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 cysteine-rich 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.

An unusual role for a mobile flavin in StaC-like indolocarbazole biosynthetic enzymes.

The indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here, we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar K(d), whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar K(d) while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD-binding motifs and the complementary StaC-10x. We find that these mutations mediate both FAD affinity and product specificity, with RebC-10x displaying higher StaC activity than StaC itself. X-ray structures of this StaC catalyst identify the substrate of StaC as 7-carboxy-K252c and suggest a unique mechanism for this FAD-dependent enzyme.

Electron-transfer rates govern product distribution in electrochemically-driven P450-catalyzed dioxygen reduction.

Developing electrode-driven biocatalytic systems utilizing the P450 cytochromes for selective oxidations depends not only on achieving electron transfer (ET) but also doing so at rates that favor native-like turnover. Herein we report studies that correlate rates of heme reduction with ET pathways and resulting product distributions. We utilized single-surface cysteine mutants of the heme domain of P450 from Bacillus megaterium and modified the thiols with N-(1-pyrene)-iodoacetamide, affording proteins that could bond to basal-plane graphite. Of the proteins examined, Cys mutants at position 62, 383, and 387 were able to form electroactive monolayers with similar E(1/2) values (-335 to -340mV vs AgCl/Ag). Respective ET rates (k(s)(o)) and heme-cysteine distances for 62, 383, and 387 are 50 s(-1) and 16Ǻ, 0.8 s(-1) and 25Ǻ, and 650 s(-1) and 19Ǻ. Experiments utilizing rotated-disk electrodes were conducted to determine the products of P450-catalyzed dioxygen reduction. We found good agreement between ET rates and product distributions for the various mutants, with larger k(s)(o) values correlating with more electrons transferred per dioxygen during catalysis.

Components of placebo effect: randomised controlled trial in patients with irritable bowel syndrome.

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. DESIGN: A six week single blind three arm randomised controlled trial. SETTING: Academic medical centre. PARTICIPANTS: 262 adults (76% women), mean (SD) age 39 (14), diagnosed by Rome II criteria for and with a score of > or =150 on the symptom severity scale. INTERVENTIONS: For three weeks either waiting list (observation), placebo acupuncture alone ("limited"), or placebo acupuncture with a patient-practitioner relationship augmented by warmth, attention, and confidence ("augmented"). At three weeks, half of the patients were randomly assigned to continue in their originally assigned group for an additional three weeks. MAIN OUTCOME MEASURES: Global improvement scale (range 1-7), adequate relief of symptoms, symptom severity score, and quality of life. RESULTS: At three weeks, scores on the global improvement scale were 3.8 (SD 1.0) v 4.3 (SD 1.4) v 5.0 (SD 1.3) for waiting list versus "limited" versus "augmented," respectively (P<0.001 for trend). The proportion of patients reporting adequate relief showed a similar pattern: 28% on waiting list, 44% in limited group, and 62% in augmented group (P<0.001 for trend). The same trend in response existed in symptom severity score (30 (63) v 42 (67) v 82 (89), P<0.001) and quality of life (3.6 (8.1) v 4.1 (9.4) v 9.3 (14.0), P<0.001). All pairwise comparisons between augmented and limited patient-practitioner relationship were significant: global improvement scale (P<0.001), adequate relief of symptoms (P<0.001), symptom severity score (P=0.007), quality of life (P=0.01). Results were similar at six week follow-up. CONCLUSION: Factors contributing to the placebo effect can be progressively combined in a manner resembling a graded dose escalation of component parts. Non-specific effects can produce statistically and clinically significant outcomes and the patient-practitioner relationship is the most robust component. TRIAL REGISTRATION: Clinical Trials NCT00065403.

Spectroscopy and electrochemistry of cytochrome P450 BM3-surfactant film assemblies.

We report analyses of electrochemical and spectroscopic measurements on cytochrome P450 BM3 (BM3) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of BM3-DDAB films on silica slides reveal the characteristic low-spin FeIII heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 FeII-CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at approximately 420 nm is present. Infrared absorption spectra of the BM3 FeII-CO complex in DDAB dispersions feature a time-dependent shift of the carbonyl stretching frequency from 1950 to 2080 cm(-1). Voltammetry of BM3-DDAB films on graphite electrodes gave the following results: FeIII/II E(1/2) at -260 mV (vs SCE), approximately 300 mV positive of the value measured in solution; DeltaS degrees (rc), DeltaS degrees , and DeltaH degrees values for water-ligated BM3 in DDAB are -98 J mol(-1) K(-1), -163 J mol(-1) K(-1), and -47 kJ mol(-1), respectively; values for the imidazole-ligated enzyme are -8 J mol(-1) K(-1), -73 J mol(-1) K(-1), and -21 kJ mol(-1). Taken together, the data suggest that BM3 adopts a compact conformation within DDAB that in turn strengthens hydrogen bonding interactions with the heme axial cysteine, producing a P420-like species with decreased electron density around the metal center.

Ginkgo biloba does not alter clearance of flurbiprofen, a cytochrome P450-2C9 substrate.

The effect of Ginkgo biloba on the activity of CYP2C9, the isoform responsible for S-warfarin clearance, was assessed in 11 healthy volunteers who received single 100-mg doses of flurbiprofen, a probe substrate for CYP2C9. Subjects also received either a standardized G biloba leaf preparation (Ginkgold, 3 doses of 120 mg) or matching placebo in a randomized, double-blind, 2-way crossover study. Mean kinetic variables for flurbiprofen with either placebo or G biloba were elimination half-life, 3.9 versus 3.5 hours; total AUC, 57 versus 55 microg/mL h; and oral clearance, 32.9 versus 31.6 mL/min. None of these differences was significant. Based on highperformance liquid chromatography analysis, each 60-mg Ginkgold tablet contained 6.6 mug of amentoflavone and 61.2 microg of quercetin, both previously identified as CYP2C9 inhibitors. These amounts were apparently too low to inhibit CYP2C9 function in vivo. The results confirm previous controlled clinical studies showing no effect of ginkgo on the kinetics or dynamics of warfarin.

Inhibition of human cytochromes P450 by components of Ginkgo biloba.

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.

Biologically active secondary metabolites from Ginkgo biloba.

Three new compounds, (7E)-2beta,3alpha-dihydroxy-megastigm-7-en-9-one (1), 3-[5,7-dihydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-yl]-4-methoxybenzoic acid (2), and 4'-O-methyl myricetin 3-O-(6-O-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside (3), were isolated from Ginkgo biloba, together with 27 known compounds. The structures of the new compounds were determined primarily from 1D- and 2D-NMR analysis. The 4-O-methylbenzoic acid structural feature at C-8 in 2 is encountered for the first time. The antioxidant activities of 29 compounds isolated from Ginkgo biloba were evaluated on intracellular reactive oxygen species in HL-60 cells. It was found that quercetin, kampferol, and tamarixetin had antioxidant activity that was approximately 3-fold greater than that of their respective glycosides and also approximately 3-fold greater than that of a standard ascorbic acid with an IC(50) at maximum effectiveness.