Crystal structure of a bifunctional deaminase and reductase from Bacillus subtilis involved in riboflavin biosynthesis

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-A resolution displayed a tetrameric ring-like structure with an extensive interface of approximately 2400 A(2)/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.     

TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment

1. Download high-res image (213KB)
2. Download full-size image

     

10′(Z),13′(E)-Heptadecadienylhydroquinone Inhibits Swarming and Virulence Factors and Increases Polymyxin B Susceptibility in Proteus mirabilis

In this study, we demonstrated that 10′(Z), 13′(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications.     

The RssAB two-component signal transduction system in Serratia marcescens regulates swarming motility and cell envelope architecture in response to exogenous saturated fatty acids

Serratia marcescens swarms at 30 degrees C but not at 37 degrees C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37 degrees C but also earlier and faster than the parent strain swarms at 30 degrees C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37 degrees C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30 degrees C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30 degrees C or 37 degrees C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.     

Triterpenoidal saponins from Hydrocotyle sibthorpioides

Oleanane-type triterpenoidal saponins, hydrocosisaponins A-F (1-6), along with a known saponin, hydrocotyloside VII (7), were isolated from Hydrocotyle sibthorpioides. Their structures were established on the basis of spectroscopic analyses including NMR spectroscopic techniques ((13)C, (1)H, COSY, HMQC, HMBC, TOCSY and NOESY). Biological evaluation established that saponins possessing four sugar units (three d-glucoses and one l-arabinose) (4-7) exhibited moderate cytotoxicity against KB, Daoy and WiDr human tumor cell lines.     

Acid-induced polymerization of the group 5 mite allergen from Dermatophagoides pteronyssinus.

House dust mites are the most important source of indoor allergens and cause allergic diseases. Our studies here suggest that the group 5 allergen from Dermatophagoides pteronyssinus (Der p 5) is monomeric at neutral pH, but forms filaments at low pH. Circular dichroism measurements show Der p 5 is a helical protein, and the protein sequence reveals Der p 5 contains coiled-coil helices. The acid-induced filament assembly could be explained in part by the high content of charged residues (40%) in the coiled-coil structure. Interestingly, some of the known Dermatophagoides allergens also contain a heptad repeat, which could potentially form coiled coils. Therefore, coiled-coil helices may be one of the common structural motifs of mite allergens that contribute to their allergenicity.     

The crystal structure of Ym1 at 1.31 A resolution

Upon nematode infection, murine peritoneal macrophages synthesize and secrete large amounts of the Ym1 protein, which is a unique functional marker for alternatively activated macrophages in T(H)2-mediated inflammatory responses. Ym1 shares significant structural similarity to the family 18 chitinases. Previously, Ym1 has been studied with respect to its carbohydrate-binding ability and glycosyl hydrolysis activity and this has led to various inconclusive interpretations. Our present co-crystallization and soaking experiments with various glucosamine or N-acetylglucosamine oligomers yield only the uncomplexed Ym1. The refined Ym1 structure at 1.31A resolution clearly displays a water cluster forming an extensive hydrogen bond network with the "active-site" residues. This water cluster contributes notable electron density to lower resolution maps and this might have misled and given rise to a previous proposal for a monoglucosamine-binding site for Ym1. A structural comparison of family 18 glycosidase (-like) proteins reveals a lack of several conserved residues in Ym1, and illustrates the versatility of the divergent active sites. Therefore, Ym1 may lack N-acetylglucosamine-binding affinity, and this suggests that a new direction should be taken to unravel the function of Ym1.     

A clinical diagnostic model for predicting influenza among young adult military personnel with febrile respiratory illness in Singapore

Introduction

Influenza infections present with wide-ranging clinical features. We aim tocompare the differences in presentation between influenza and non-influenzacases among those with febrile respiratory illness (FRI) to determinepredictors of influenza infection.

Methods

Personnel with FRI (defined as fever≥37.5°C, with cough or sorethroat) were recruited from the sentinel surveillance system in theSingapore military. Nasal washes were collected, and tested using theResplex II and additional PCR assays for etiological determination.Interviewer-administered questionnaires collected information on patientdemographics and clinical features. Univariate comparison of the variousparameters was conducted, with statistically significant parameters enteredinto a multivariate logistic regression model. The final multivariate modelfor influenza versus non-influenza cases was used to build a predictiveprobability clinical diagnostic model.

Results

821 out of 2858 subjects recruited from 11 May 2009 to 25 Jun 2010 hadinfluenza, of which 434 (52.9%) had 2009 influenza A (H1N1), 58(7.1%) seasonal influenza A (H3N2) and 269 (32.8%) influenzaB. Influenza-positive cases were significantly more likely to present withrunning nose, chills and rigors, ocular symptoms and higher temperature, andless likely with sore throat, photophobia, injected pharynx, andnausea/vomiting. Our clinical diagnostic model had a sensitivity of65% (95% CI: 58%, 72%), specificity of69% (95% CI: 62%, 75%), and overall accuracy of68% (95% CI: 64%, 71%), performing significantlybetter than conventional influenza-like illness (ILI) criteria.

Conclusions

Use of a clinical diagnostic model may help predict influenza better than theconventional ILI definition among young adults with FRI.