The structure of glucose‐1‐phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA‐type enzymes

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six‐month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure‐based drug design is the enzyme RmlA, a glucose‐1‐phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l ‐rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X‐ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP‐glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP‐glucose complex, only a single magnesium ion was observed. Importantly, for RmlA‐type enzymes with known three‐dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide‐linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure‐based drug design. In the past, molecular modeling procedures have been employed to derive a three‐dimensional model of the M. tuberculosis RmlA for drug design. The X‐ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.     

Molecular architectures of Pen and Pal: Key enzymes required for CMP‐pseudaminic acid biosynthesis in Bacillus thuringiensis

Bacillus thuringiensis is a soil‐dwelling Gram positive bacterium that has been utilized as a biopesticide for well over 60 years. It is known to contain flagella that are important for motility. One of the proteins found in flagella is flagellin, which is post‐translationally modified by O‐glycosylation with derivatives of pseudaminic acid. The biosynthetic pathway for the production of CMP‐pseudaminic acid in B. thuringiensis, starting with UDP‐N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc), requires seven enzymes. Here, we report the three‐dimensional structures of Pen and Pal, which catalyze the first and second steps, respectively. Pen contains a tightly bound NADP(H) cofactor whereas Pal is isolated with bound NAD(H). For the X‐ray analysis of Pen, the site‐directed D128N/K129A mutant variant was prepared in order to trap its substrate, UDP‐GlcNAc, into the active site. Pen adopts a hexameric quaternary structure with each subunit showing the bilobal architecture observed for members of the short‐chain dehydrogenase/reductase superfamily. The hexameric quaternary structure is atypical for most members of the superfamily. The structure of Pal was determined in the presence of UDP. Pal adopts the more typical dimeric quaternary structure. Taken together, Pen and Pal catalyze the conversion of UDP‐GlcNAc to UDP‐4‐keto‐6‐deoxy‐l ‐N‐acetylaltrosamine. Strikingly, in Gram negative bacteria such as Campylobacter jejuni and Helicobacter pylori, only a single enzyme (FlaA1) is required for the production of UDP‐4‐keto‐6‐deoxy‐l ‐N‐acetylaltrosamine. A comparison of Pen and Pal with FlaA1 reveals differences that may explain why FlaA1 is a bifunctional enzyme whereas Pen and Pal catalyze the individual steps leading to the formation of the UDP‐sugar product. This investigation represents the first structural analysis of the enzymes in B. thuringiensis that are required for CMP‐pseudaminic acid formation.     

Chloroplast Signaling Gates Thermotolerance in Arabidopsis

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Liquified Petroleum Gas (LPG) Supply and Demand for Cooking in Northern Ghana

Like many other countries, Ghana relies on biomass (mainly wood and charcoal) for most of its cooking needs. A national action plan aims to expand liquefied petroleum gas (LPG) access to 50% of the country’s population by 2020. While the country’s southern urban areas have made progress toward this goal, LPG use for cooking remains low in the north. The aim of this cross-sectional study was to characterize the current state of the LPG market in this area and examine opportunities and barriers to scale up LPG adoption. We interviewed 16 LPG suppliers (stove, cylinder, and fuel vendors) as well as 592 households in the Kassena-Nankana Districts (KND) of Ghana. We find large rural–urban differences in LPG uptake: less than 10% of rural households own LPG stoves compared with over half of urban households. Awareness of LPG is high across the region, but accessibility of fuel supply is highly limited, with just one refilling station located in the KND. Affordability is perceived as the main barrier to LPG adoption, and acceptability is also limited by widespread concerns about the safety of cooking with LPG. Transitioning to a cylinder recirculation model, and providing more targeted subsidies and credit options, should be explored to expand access to cleaner cooking in this region.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

The Genome Sequence DataBase (GSDB): meeting the challenge of genomic sequencing.

The genome sequence database (GSDB) is a complete, publicly available relational database of DNA sequences and annotation maintained by the National Center for Genome Resources (NCGR) under a Cooperative Agreement with the US Department of Energy (DOE). GSDB provides direct, client- server access to the database for data contributions, community annotation and SQL queries. The GSDB Annotator, a multi-platform graphic user interface, is freely available. Automatically updated relational replicates of GSDB are also freely available.     

Enzymatic basis for sialyl-Tn expression in human colon cancer cells

Sialyl-Tn antigen (SA2-6 GalNAc-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R 3-Gal-transferase (core 1 3-Gal-transferase) was lacking. Core 1 3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc1-6 (Gal1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 3-Gal-transferase activity. The lack of core 1 3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.