The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.     

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.     

Mutational analysis and characterization of nocardicin C-9' epimerase

The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic beta-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a d-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5'-phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the l-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9' epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of beta-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.     

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II

Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (K(T(DS)) 1-5.8 microm; K(T(H)) 0.02-0.2 microm) than to HCII (K(HCII(DS)) 236-291 microm; K(HCII(H)) 25-35 microm), favoring formation of T.GAG over HCII.GAG complexes as intermediates for T.GAG.HCII complex assembly. At [GAG] < K(HCII(GAG)) the GAG and HCII concentration dependences of the first-order inactivation rate constants (k(app)) were hyperbolic, reflecting saturation of T.GAG complex and formation of the T.GAG.HCII complex from T.GAG and free HCII, respectively. At [GAG] > K(HCII(GAG)), HCII.GAG complex formation caused a decrease in k(app). The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T.GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with K(MzT(-F1)(GAG)) 10 and 20 microm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T.DS and MzT(-F1).DS interactions were saturable at DS concentrations substantially below K(HCII(DS)), consistent with DS bridging T.DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex.     

Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Autosomal dominant FEVR is genetically heterogeneous, but its principal locus, EVR1, is on chromosome 11q13-q23. The gene encoding the Wnt receptor frizzled-4 (FZD4) was recently reported to be the EVR1 gene, but our mutation screen revealed fewer patients harboring mutations than expected. Here, we describe mutations in a second gene at the EVR1 locus, low-density-lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor. This finding further underlines the significance of Wnt signaling in the vascularization of the eye and highlights the potential dangers of using multiple families to refine genetic intervals in gene-identification studies.     

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.