DADH catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding alpha-keto acids and ammonia. Here we report the first X-ray crystal structures of DADH at 1.06 A resolution, and its complexes with iminoarginine (DADHred/iminoarginine) and iminohistidine (DADHred/iminohistidine) at 1.30 A resolution. The DADH crystal structure comprises an unliganded conformation and a product-bound conformation, which is almost identical to the DADHred/iminoarginine crystal structure. The active site of DADH was partially occupied with iminoarginine product (30% occupancy) that interacts with Tyr53 in the minor conformation of a surface loop. This flexible loop forms an "active site lid", similar to those seen in other enzymes, and may play an essential role in substrate recognition. The guanidinium side chain of iminoarginine forms a hydrogen bond interaction with the hydroxyl of Thr50 and an ionic interaction with Glu87. In the structure of DADH in complex with iminohistidine, two alternate conformations were observed for iminohistidine where the imidazole groups formed hydrogen bond interactions with the side chains of His48 and Thr50 and either Glu87 or Gln336. The different interactions and very distinct binding modes observed for iminoarginine and iminohistidine are consistent with the 1000-fold difference in kcat/Km values for D-arginine and D-histidine. Comparison of the kinetic data for the activity of DADH on different D-amino acids and the crystal structures in complex with iminoarginine and iminohistidine establishes that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity.

Heat shock protein (Hsp) 40s play essential roles in cellular processes by cooperating with Hsp70s. Hsp40s recognize non-native polypeptides, deliver these peptides to Hsp70s and stimulate the ATPase activity of Hsp70s to facilitate the correct folding of the polypeptides. We have determined the crystal structures of the C-terminal peptide-binding domain of human Hsp40 Hdj1 (CTD) and of its complex with the C-terminal octapeptide of human Hsp70, ^634'GPTIEEVD^641'. CTD exists as a twisted, horseshoe-shaped homodimer. The protomer consists of the two domains I and II with similar topologies. The octapeptides are located in two sites, 1 and 2, of domain I. In site 1, the octapeptide forms an anti-parallel beta-sheet with CTD. The negatively charged residues of the EEVD motif in the octapeptide form electrostatic interactions with the positively charged Lys residues of CTD. The Ile side-chain of the octapeptide fits into the narrow concave formed by the hydrophobic residues of CTD. In site 2, the octapeptide also forms an anti-parallel beta-sheet with CTD and the EEVD motif forms electrostatic interactions. The side-chains of Pro and Ile of the octapeptide interact with the hydrophobic surface region of CTD site 2, which is broader and shallower than the concave binding region of site 1. This region seems to be capable of binding hydrophobic side-chains that are bulkier than the Ile side-chain. From these structural characteristics, site 1 represents the peptide-recognition site of the Hsp70 C-terminus, whereas site 2 presumably represents the binding site toward non-native polypeptides.

Cry11Ba produced by <i>Bacillus thuringiensis</i> subsp. jegathesan is an active toxin for larvae of the mosquito <i>Anopheles gambiae</i>. A 106-kDa aminopeptidase N, called AgAPN2, was previously identified as a Cry11Ba receptor in <i>A. gambiae</i>. A 70-kDa fragment of AgAPN2 expressed in Escherichia coli binds Cry11Ba with high affinity (Kd = 6.4 nM) and inhibits Cry11Ba activity by 98% in bioassays [Zhang et al. (2008) Biochemistry 47, 11263-72]. To identify regions involved in toxicity, we truncated the 70-kDa APN fragment into peptides of 28- and 30-kDa ta and tb respectively, and tested their abilities to mediate toxicity and bind Cry11Ba. While AgAPN2ta reduced Cry11Ba toxicity by 85%, AgAPN2tb showed a significant enhancement effect on Cry11Ba toxicity. The purified peptides showed evidence of structural folding and bound the same site(s) on Cry11Ba with high affinity. The inhibitory AgAPN2ta blocked Cry11Ba binding to brush border membrane vesicles (BBMV) of A. gambiae whereas the toxicity enhancing AgAPN2tb increased Cry11Ba binding on BBMV. A deletion at the N-terminus (336S - P420) of AgAPN2ta significantly reduced AgAPN2ta binding to Cry11Ba and its inhibitory effect. Deletion of the central region (676I - W760) of AgAPN2tb eliminated its increased toxin binding and toxicity enhancement effect without affecting Cry11Ba binding. A 'bridge' model is proposed for AgAPN2tb action whereby the peptide binds Cry11Ba and vectors it to sites on the larval midgut.

Carbonyl reduction is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics. Carbonyl reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, has been poorly characterized so far and the regulation of its expression is completely unknown. Here, we show that CBR3 expression is regulated via Nrf2, a key regulator in response to oxidative stress. In human cancer cell lines, CBR3 mRNA was expressed differentially ranging from very high (A549; lung) to very low (HT-29; colon, HepG2; liver) levels. CBR3 protein was highly expressed in SW-480 (colon) cells, but was absent in HCT116 (colon) and HepG2 cells. CBR3 mRNA was inducible in HT-29 cells by Nrf2-agonists (sulforaphane [SUL; 7-fold], diethylmaleate [DEM; 4-fold]) or hormone receptor ligand Z-guggulsterone (5-fold), respectively. Aryl hydrocarbon receptor agonist B[k]F failed to induce CBR3 mRNA after 8 h of incubation but elevated CBR3 levels after 24 h, most likely mediated by B[k]F-metabolites that can activate Nrf2-signaling. Inhibition of Nrf2-activating upstream kinase MEK/ERK by PD98059 decreased DEM-mediated induction of CBR3 mRNA. Proteasome inhibitors MG-132 (5 microM) and bortezomib (50 nM) dramatically increased CBR3 mRNA, obviously due to the increase in Nrf2 protein. While siRNA-mediated knock-down of Nrf2 led to a decrease of CBR3 mRNA in A549 cells (30% of control), Keap1 knock-down increased CBR3 mRNA expression in HepG2 (9.3-fold) and HT-29 (2.7-fold) cells. Here, we provide for the first time evidence that human CBR3 is a new member of the Nrf2-gene battery.

Meprin alpha and beta, zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like IL-1beta, IL-18 or TGF-alpha. Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified yet. In this work we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K_i (inhibition constant) of 4.2*10^-5 M for meprin alpha and a K_i of 1.1*10^-6 M for meprin beta. This correlated with data obtained for a fetuin-A homolog from carp (nephrosin inhibitor) which revealed a potent meprin alpha and beta inhibition (residual activities of 27% and 22%, respectively) at a carp fetuin concentration of 1.5*10^-6 M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin alpha activity was 40 times weaker than meprin beta activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin alpha (K_i = 8.5*10^-6 M), but, interestingly, not for meprin beta. Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broaden our understanding of the proteolytic network in plasma.

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labelling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage lambda (lambda lysozyme) at pH 5.6, 20 degrees C. The first step in the folding of lambda lysozyme occurs very rapidly (tau < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure, but only weak protection from 1H/2H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with tau = 0.15 +/- 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (tau = 5 +/- 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that lambda lysozyme is a highly dynamic protein, with relatively low conformational stability (DeltaG degrees N-U = 25 +/- 2 kJ*mol-1).

A reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer coupled IANUS (<b>I</b>nduced org<b>AN</b>ization of str<b>U</b>cture by matrix-assisted togethernes<b>S</b>) peptide array. Here we report that parvulin 10 (Par10), an abundant <i>Escherichia coli</i> (<i>E. coli</i>) peptidyl prolyl <i>cis/trans</i> isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative <i>E. coli</i> strain showed increased sensitivity towards hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of ¹⁵N-labeled Par10 in the presence of AhpC^115-132 peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10/AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline <i>cis</i> and <i>trans</i> isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as substrate for Par10 enzymatic action. Based on these data we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress.

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase and a major controller of cell growth. In cells, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. The mTORC1 complex can phosphorylate 4EBP1 and S6K1, two key regulators of translation initiation, whereas mTORC2 phosphorylates AKT1, an event required for AKT1 activation. Here, we expressed and purified human mTORC1 and mTORC2 from HEK-293 cells using FLAG-M2 affinity chromatography. Western blotting analysis using phospho-specific antibodies indicated that recombinant mTORC1 and mTORC2 exhibit distinct substrate preferences in vitro, consistent with their roles in cells. To better understand the enzymatic properties of mTOR alone and mTOR in its complex form, steady-state kinetic profiles of truncated mTOR containing kinase domain (residues 1360-2549) and mTORC1 were determined. The results revealed that mTORC1 is catalytically less active than truncated mTOR, as evidenced by 4.7- and 3.1-fold decrease in catalytic efficiency, kcat/Km, for ATP and 4EBP1, respectively. We also found that truncated mTOR undergoes autophosphorylation through an intramolecular mechanism. Mass spectrometric analysis identified two novel mTOR autophosphorylation sites, Ser2454 and one of Thr2473/Thr2474, in addition to the previously reported Ser2481 site. Truncated mTOR and mTORC1 were completely inhibited by ATP competitive inhibitors PI103 and BEZ235, while partially inhibited by rapamycin/FKBP12 in a noncompetitive fashion toward ATP. All inhibitors tested exhibited similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain. Our studies presented here provide the first detailed kinetic studies of a recombinant mTOR complex.

Nanodiscs are an example of discoidal nanoscale lipid/protein particles that have been extremely useful for the biochemical and biophysical characterization of membrane proteins. They are discoidal lipid bilayer fragments encircled and stabilized by two amphipathic helical proteins named membrane scaffolding protein (MSP), ~10 nm in size. Nanodiscs are homogeneous, easily prepared with reproducible success, amenable to preparations with a variety of lipids, and stable under a range of temperatures. Here we present solid-state NMR (SSNMR) studies on lyophilized, rehydrated POPC Nanodiscs prepared with uniformly 13C, 15N-labeled MSP1D1 (Delta1-11 truncated MSP). Under these conditions, by SSNMR we directly determine the gel-to-liquid crystal lipid phase transition to be at 3 +/- 2 degrees C. Above this phase transition, the lipid 1H signals have slow transverse relaxation, enabling filtering experiments as previously demonstrated for lipid vesicles. We incorporate this approach into two- and three-dimensional heteronuclear SSNMR experiments to examine the MSP1D1 residues interfacing with the lipid bilayer. These 1H-13C and 1H-13C-13C correlation spectra are used to identify and quantify the number of lipid-correlated and solvent-exposed residues by amino acid type, which furthermore is compared with molecular dynamics studies of MSP1D1 in Nanodiscs. This study demonstrates the utility of SSNMR experiments with Nanodiscs for examining lipid-protein interfaces and has important applications for future structural studies of membrane proteins in physiologically relevant formulations.

Aspergillus fumigatus Af293 is a known producer of quinazoline natural products including the antitumor fumiquinazolines, of which the simplest member is fumiquinazoline F (FQF) with a 6-6-6 tricyclic core derived from anthranilic acid, tryptophan, and alanine. FQF is the proposed biological precursor to fumiquinazoline A (FQA) where the pendant indole side chain has been modified via oxidative coupling of an additional molecule of alanine, yielding a fused 6-5-5 imidazoindolone. We recently identified fungal anthranilate-activating non-ribosomal peptide synthetase (NRPS) domains through bioinformatics approaches. One domain previously identified is part of the trimodular NRPS Af12080, which we predict is responsible for FQF formation. We now show that two adjacent A. fumigatus ORFs, a monomodular NRPS Af12050 and a flavoprotein Af12060, are necessary and sufficient to convert FQF to FQA. Af12060 oxidizes the 2',3'-double bond of the indole side chain of FQF, and the three-domain NRPS Af12050 activates L-Ala as the adenylate, installs it as the pantetheinyl thioester on its carrier protein domain and acylates the oxidized indole for subsequent intramolecular cyclization to create the 6-5-5 imidazolindolone of FQA. This work provides experimental validation of the fumiquinazoline biosynthetic cluster of A. fumigatus A293, and describes an oxidative annulation biosynthetic strategy likely shared among several classes of polycyclic fungal alkaloids.

alpha-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalising the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed and more water soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity towards Jurkat cells. On the basis of gel filtration data, crosslinking experiments with glutaraldehyde and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation and it interacts with the fatty acid mostly through its alpha-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.

The central region of the LacI N-subdomain monomer*monomer interface includes residues K84, V94, V95, V96, S97, and M98. The side chains of these residues line the beta-strands at this interface and interact to create a network of hydrophobic, charged, and polar interactions that significantly rearranges in different functional states of LacI. Prior work showed that converting K84 to an apolar residue or converting V96 to an acidic residue impedes the allosteric response to inducer. Thus, we postulated that a disproportionate number of substitutions in this region of the monomer*monomer interface would alter the complex features of the LacI allosteric response. To explore this hypothesis, acidic, basic, polar, and apolar mutations were introduced at positions 94-98. Despite their varied locations along the beta-strands that flank the interface, ~70% of the mutations impact allosteric behavior, with the most significant effects found for charged substitutions. Of note, many of the LacI variants with minor functional impact exhibited altered stability to urea denaturation. The results confirm the critical role of amino acids 94-98 and indicate that this N-subdomain interface forms a primary pathway in LacI allosteric response.

In gram negative bacteria like E. coli the ExbB-ExbD-TonB protein complex is anchored to the cytoplasmic membrane and is involved in energization of outer membrane transport. Outer membrane proteins catalyze energy-coupled transport of scarce nutrients. Energy is derived from the proton motive force of the cytoplasmic membrane which is transferred through ExbB-ExbD-TonB to the outer membrane transporters. Earlier studies showed that ExbB is the most abundant protein of the ExbB-ExbD-TonB complex and stabilizes TonB and ExbD. To advance understanding of the role of ExbB in the membrane organization of the ExbB-ExbD-TonB complex, His-tagged ExbB was solubilized with decyl maltoside and purified to electrophoretic homogeneity. Its size and shape was determined by blue-native gel electrophoresis, size exclusion chromatography, transmission electron microscopy and small angle X-ray scattering. Decyl maltoside bound to ExbB was quantified by the Anthrone method that determines the sugar moiety of decyl maltoside. The results obtained with the four methods consistently indicated that isolated ExbB adopts a stable homooligomer with 4-6 monomers. We propose that the ExbB homooligomer forms a platform on which ExbD and TonB are assembled to form the energy-transducing complex in the cytoplasmic membrane.

The regulated activation of NF-kappaB by antigen receptor signaling is required for normal B and T lymphocyte activation during the adaptive immune response. Dysregulated NF-kappaB activation is associated with several types of lymphoma, including Diffuse Large B Cell Lymphoma (DLBCL). During normal antigen receptor signaling, the multidomain scaffold protein CARD11 undergoes a transition from a closed, inactive state to an open, active conformation that recruits several signaling proteins into a complex, leading to IKK kinase activation. This transition is regulated by the CARD11 Inhibitory Domain (ID), which participates in intramolecular interactions that prevent cofactor binding to CARD11 prior to signaling, but which is neutralized after receptor engagement by phosphorylation. Several oncogenic CARD11 mutations have been identified in DLBCL that enhance activity and that are mostly found in the Coiled-coil domain. However, the mechanisms by which these mutations cause CARD11 hyperactivity and spontaneous NF-kappaB activation are poorly understood. In this report, we provide several lines of evidence that oncogenic mutations F123I and L225LI induce CARD11 hyperactivity by disrupting autoinhibition by the CARD11 ID. These mutations disrupt ID-mediated intramolecular interactions, ID-dependent inhibition, and bypass the requirement for ID phosphorylation during T cell receptor signaling. Intriguingly, these mutations selectively enhance the apparent affinity of CARD11 for Bcl10, but not for other signaling proteins that are recruited to CARD11 in an ID-dependent manner during normal antigen-receptor signaling. Our results establish a mechanism that explains how DLBCL-associated mutations in CARD11 can initiate spontaneous, receptor-independent activation of NF-kappaB.

The Wilson disease protein (ATP7B) is a copper-transporting member of the P-type ATPase superfamily, which plays a central role in copper homeostasis and interacts with the copper chaperone Atox1. The N-terminus of ATP7B is comprised of six copper-binding domains (WCBDs), each capable of binding one copper atom in the +1 oxidation state. To better understand the regulatory effect of copper binding to these domains, we have performed NMR characterization of WCBD4-6 (domains 4-6 of ATP7B). 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is no dramatic change in the dynamic properties of this three-domain construct; the linker between domains 4 and 5 remains flexible, domains 5 and 6 do not form a completely rigid dimer but rather have some flexibility with respect to each other, and there is minimal change in the relative orientation of the domains in the two states. We also show that, contrary to previous reports, the protein-protein interaction between Atox1 and the copper-binding domains takes place even in the absence of copper. Comparison of apo and Cu(I)-bound spectra of WCBD1-6 show that binding of Cu(I) does not induce the formation of a unit that tumbles as a single entity, consistent with our results for WCBD4-6. We propose that copper transfer to and between the N-terminal domains of the Wilson Cu-ATPase occur via protein interactions that are facilitated by the flexibility of the linkers and the motional freedom of the domains with respect to each other.

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine-S-sulfoxide (Met-SO), and methionine-R-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB or both proteins, had normal levels of mitochondria, but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Raltegravir is an FDA approved inhibitor directed against human immunodeficiency virus type 1 (HIV-1) integrase (IN). In this study, we investigated the mechanisms associated with multiple strand transfer inhibitors capable of inhibiting concerted integration by HIV-1 IN. The results show raltegravir, elvitegravir, MK-2048, RDS 1997, and RDS 2197 all appear to encompass a common inhibitory mechanism by modifying IN-viral DNA interactions. These structurally different inhibitors bind to and inactivate the synaptic complex, an intermediate in the concerted integration pathway in vitro. The inhibitors physically trap the synaptic complex thereby prevent target DNA binding and thus concerted integration. The efficiency of a particular inhibitor to trap the synaptic complex observed on native agarose gels correlated with its potency for inhibiting the concerted integration reaction, defined by IC₅₀ values for each inhibitor. At low nM concentrations (<50 nM), raltegravir displayed a time-dependent inhibition of concerted integration, a property associated with slow-binding inhibitors. Studies of raltegravir resistant IN mutants N155H and Q148H without inhibitors demonstrated that their capacity to assembly the synaptic complex and promote concerted integration were similar to their reported virus replication capacities. The concerted integration activity of Q148H showed a higher cross-resistance to raltegravir than observed with N155H providing evidence as to why Q148H pathway with secondary mutations is the predominant pathway upon prolong treatment. Notably, MK-2048 is equally potent against wild type IN and raltegravir resistant IN mutant N155H suggesting this inhibitor may bind similarly within their drug-binding pockets.

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular-weight thiol in <i>Bacillus anthracis</i>, <i>Staphylococcus aureus</i>, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in <i>B. anthracis</i>, which combine to produce alpha-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the <i>Ba</i>BshA glycosyltransferase (GlcNAc-malate) and the <i>Ba</i>BshB deacetylase (GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the <i>Ba</i>BshA-UDP-malate ternary complex, determined in this work at 3.3 A resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other alpha-hydroxyacids, as acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-D-<i>myo</i>-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding <i>Ba</i>BshA forms. A mutant strain of <i>B. anthracis</i> deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This <i>B. anthracis bshA</i> locus (BA1558) has been identified in a transposon site hybridization study as required for growth, sporulation, or germination [Day, W.A., Jr., Rasmussen, S.L., Carpenter, B.M., Peterson, S.N., and Friedlander, A.M. (2007) <i>J. Bacteriol. 189</i>, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for development of novel antimicrobials with broad spectrum activity against Gram-positive pathogens like <i>B. anthracis</i>. The metabolites that function in thiol redox buffering and homeostasis in <i>Bacillus</i> are not well understood, and we present a composite picture based on this and other recent work.

beta-amyloid (Abeta) is the main protein component of the amyloid plaques associated with Alzheimer's disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type transthyretin (TTR) amyloid deposits are linked to senile systemic amyloidosis, a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from toxic effects of Abeta deposition [Stein, T.D. and Johnson, J.A. (2002) J. Neurosci. 22: 7380-7388]. We examined the interaction of Abeta with wt TTR as well as two mutants: F87M/L110M, engineered to be a stable monomer, and T119M, a naturally occurring mutant with higher tetrameric stability than wildtype. Based on enzyme-linked immunoassays as well as crosslinking experiments, we conclude that Abeta monomers bind more strongly to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Abeta aggregates rather than Abeta monomers. Through tandem mass spectrometry analysis of crosslinked TTR-Abeta fragments, we identified the A strand, in the inner beta-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Abeta association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Abeta interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation, TTR monomers decidedly arrest Abeta aggregate growth. These data provide important new insights into the nature of TTR-Abeta interactions. Such interactions may regulate TTR-mediated protection against Abeta toxicity.

Continuous oxidative damage inflicted on DNA produces 7,8-dihydro-8-oxoguanine (8-oxoG), a commonly occurring lesion that can potentially cause cancer by producing G T transversions during DNA replication. Mild oxidation of 8-oxoG leads to the formation of hydantoins, specifically guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), which are 100% mutagenic because they code almost exclusively for the insertion of dAMP and dGMP (coding for G T and G C transversions, respectively). The wild type (wt) pol family DNA polymerase from bacteriophage RB69 (RB69pol) inserts dAMP and dGMP with low efficiency when situated opposite Gh. In contrast, the RB69pol Y567A mutant inserts both of these dNMPs opposite Gh with > 100-fold higher efficiency than wt. We now report the crystal structure of the "closed" pre-insertion complex for the Y567A mutant with dATP opposite a templating Gh (R-configuration) in a 13/18mer primer-template (P/T) at 2.0 A resolution. The structure data reveals that the Y to A substitution provides the Nascent base-pair Binding Pocket (NBP) with the flexibility to accommodate Gh by allowing G568 to move toward the minor groove in the P/T. Thus, Gh is rejected as a templating base by wt RB69pol because G568 is inflexible, preventing Gh from pairing with the incoming dATP or dGTP base.