The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone.

The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9-Å resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) 2 catalyze the acetyl-CoA-dependent N-acetylation of primary aromatic amines and hydrazines, as well as the O-acetylation of their N-hydroxylated metabolites, thereby influencing the biological activity and toxicity of this class of chemicals (1-3). NAT enzyme activity therefore plays an important role in determining the duration of action and pharmacokinetics of aromatic amine-containing drugs used in clinical therapy, as well as in influencing the balance between detoxification and metabolic activation of aromatic amine procarcinogens (4). In the latter regard, N-acetylation reactions are considered to be protective, because the resulting arylacetamide derivatives are chemically stable, whereas O-acetylation of hydroxylamines produces acetoxy esters that can spontaneously decompose to electrophilic, DNA-binding nitrenium ions (5). Thus a better understanding of the structural features of NATs that contribute to their relative abilities to catalyze such reactions for various amine substrates may be of considerable predictive value in optimizing drug development and biomedical toxicology.Two NATs, NAT1 and NAT2, have been annotated in the human genome. Each of these enzymes is genetically variable in human populations, with variable activity of NAT2 being responsible for the classic acetylation polymorphism that was discovered over half a century ago with the advent of isoniazid therapy for tuberculosis (6 -8). In addition to isoniazid, the disposition of several other therapeutically useful drugs is affected by defective NAT2 function, including hydralazine, phenelzine, procainamide, and some of the sulfonamide antibacterials. NAT1 is highly homologous to NAT2 yet kinetically distinct, such that certain aromatic amines (such as isoniazid and sulfamethazine) are preferentially acetylated by NAT2, whereas others (such as p-aminosalicylic acid and p-aminobenzoic acid) are selective substrates for NAT1. NAT1 is also genetically variable in human populations. Numerous epidemiological studies have reported associations between both NAT1 and NAT2 variation and the occurrence of cancers related to exposure to aromatic...

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5D23 and its complexes with unmodified, mono-, di-and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes.

Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with mannose moieties by a "click" chemistry approach, are able to efficiently inhibit E. coli type 1 fimbriae-mediated adhesion to eukaryotic cells with relative inhibitory potency (RIP) of as high as 9259 (bladder cell adhesion assay), which is unprecedented when compared with RIP values previously reported for alternate multivalent mannose-functionalized nanostructures designed to inhibit E. coli adhesion. Also remarkable is that these novel mannose-modified NDs reduce E. coli biofilm formation, a property previously not observed for multivalent glyco-nanoparticles and rarely demonstrated for other multivalent or monovalent mannose glycans. This work sets the stage for the further evaluation of these novel NDs as an anti-adhesive therapeutic strategy against E. coli-derived infections.

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by ␤-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2-to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 Å of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.In eubacteria and their endosymbiotic descendants (the plastids of plants and apicomplexan parasites) fatty acids are produced by what is known as the type II fatty acid biosynthetic pathway (1-3). The steps in this pathway are catalyzed by a universal set of enzymes, each encoded by a separate gene, that have been most closely studied in the model organism Escherichia coli (4, 5). The growing acyl chain is shuttled between the pathway enzymes attached to the 4Ј-phosphopantetheine prosthetic group of a dedicated carrier protein, ACP.1 This system contrasts with the type I fatty acid biosynthesis system that exists in metazoans, where multifunctional polypeptide chains (6) encode all activities in chain initiation and elongation. The intermediates in the type I system are shuffled from one catalytic site to another without being released from the complex. In light of the profound differences in these two systems, enzymes of the type II pathway have emerged as attractive targets for the development of novel antimicrobial or antiparasitic agents (2,7,8). The core feature of the type II pathway is the fatty acid elongation cycle, which extends the fatty acid chain by two carbons in each round. There are four steps in the cycle, and the proteins involved in E. coli are 1) condensation of malonyl-ACP with acyl-ACP, catalyzed by the FabB-and FabF-condensing enzymes, 2) reduction of the ␤-keto moiety by the NADPH-dependent FabG re...

Low dose perioperative intravenous lidocaine after total hip arthroplasty offers no beneficial effect on postoperative analgesia and does not modify pressure and tactile pain thresholds.

dBrucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ⌬btaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ⌬btaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.

Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure–function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation.