M. C. J. Wilce

Crystal structure of a theta-class glutathione transferase.

Glutathione S‐transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Cytosolic GSTs have been grouped into four evolutionary classes for which there are representative crystal structures of three of them. Here we report the first crystal structure of a theta‐class GST. So far, all available GST crystal structures suggest that a strictly conserved tyrosine near the N‐terminus plays a critical role in the reaction mechanism and such a role has been convincingly demonstrated by site‐directed mutagenesis. Surprisingly, the equivalent residue in the theta‐class structure is not in the active site, but its role appears to have been replaced by either a nearby serine or by another tyrosine residue located in the C‐terminal domain of the enzyme.

Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

Crystal structure of a UDP-glucose-specific glycosyltransferase from a Mycobacterium species.

Glycosyltransferases (GTs) are a large and ubiquitous family of enzymes that specifically transfer sugar moieties to a range of substrates. Mycobacterium tuberculosis contains a large number of GTs, many of which are implicated in cell wall synthesis, yet the majority of these GTs remain poorly characterized. Here, we report the high resolution crystal structures of an essential GT (MAP2569c) from Mycobacterium avium subsp. paratuberculosis (a close homologue of Rv1208 from M. tuberculosis) in its apo- and ligand-bound forms. The structure adopted the GT-A fold and possessed the characteristic DXD motif that coordinated an Mn2+ ion. Atypical of most GTs characterized to date, MAP2569c exhibited specificity toward the donor substrate, UDP-glucose. The structure of this ligated complex revealed an induced fit binding mechanism and provided a basis for this unique specificity. Collectively, the structural features suggested that MAP2569c may adopt a “retaining” enzymatic mechanism, which has implications for the classification of other GTs in this large superfamily.

HOMOLOGY MODEL FOR THE HUMAN GSTT2 THETA CLASS GLUTATHIONE TRANSFERASE

A tertiary model of the human GSTT2 Theta class glutathione transferase is presented based on the recently solved crystal structure of a related thetalike isoenzyme from Lucilia cuprina. Although the N‐terminal domains are quite homologous, the C‐terminal domains share less than about 20% identity. The model is used to consolidate the role of Ser 11 in the active site of the enzyme as well as to identify other residues and mechanisms of likely catalytic importance. The T2 subfamily of theta class enzymes have been shown to inactivate reactive sulfate esters arising from arylmethanols. A possible reaction pathway involving the conjugation of glutathione with one such sulfate ester, 1‐menaphthyl‐sulfate, is described. It is also proposed that the C‐terminal region of the enzyme plays an important role in allowing substrate access to the active site. Proteins 27:118–130

Crystallization and preliminary X-ray diffraction studies of a glutathione S-Transferase from the Australian sheep blowfly, Lucilia cuprina

Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.

Crystallization and preliminary X-ray analysis of a bacterial glutathione transferase

Crystals of a bacterial glutathione S-transferase from Proteus mirabilis have been grown from polyethylene glycol by the hanging-drop vapour-diffusion method. Successful crystallization required the presence of the substrate glutathione. The crystals belong to the tetragonal space group P4 with cell dimensions a = b = 90.9 and c = 117.3 A. They contain between three and six monomers in the asymmetric unit and diffract to beyond 2.3 A resolution.

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor

We have selected the mediterranean shrub Euphorbia characias as an experimental model to study the complexity of plant latex chemistry. Latex is a mixture with diversified composition, that includes alkaloids, terpenoid compounds, polymeric substances such as resins and gums, starch, oil and a large number of proteins and enzymatic activities. The aim of the present study is to contribute to the knowledge of this plant product evaluating the antioxidant properties of extracts of E. characias and searching for polymeric substances as natural rubber. We analyzed different extracts from the latex of E. characias and performed a new extraction method (involving the use of trichloacetic acid, TCA) that turned out to be easier, faster and higher reproducible if compared to common extraction methods involving organic solvents like methanol, ethanol, and petroleum ether/methanol. TCA extract of E. characias latex exhibits antioxidant activities determined as total content of free-radical scavenging, polyphenols and total flavonoids. GC-MS analysis confirms the presence of several compound identified as antioxidant molecules. E. characias latex contains a natural rubber. The optimum rubber extraction is achieved with acetic acid followed by cyclohexane/ethanol treatment. The rubber content was shown to be 14% (w/v) of the E. characias latex and the gel content is 2.5% of the rubber weight. E. characias natural rubber showed a molecular weight of 93000 and Mw/Mn of 2.9. On the basis of H NMR, C NMR and FT-IR spectroscopy, the structure of this rubber can be identified as cis-1,4-polyisoprene. This study was partially supported by a grant from Regione Autonoma della Sardegna, Progetti di ricerca di base CRP2-22.Session 1—Maternal Medicine 1. INCIDENCE OF SUPPLEMENTAL POLYDRUG USE IN IRISH PREGNANT WOMEN ON METHADONE MAINTENANCE PROGRAMME Akhter P, Coulter-Smith S, Lee J, Brennan M, Clarke T, Geary M; Dublin, Ireland. 3. MAXIMAL EXERCISE TESTING CAN BE SAFELY USED TO ASSESS CARDIOVASCULAR RESERVE IN PREGNANCY Barker D, Mason G, Schlosshan D, McLoughlin H, Blackburn M, Simpson N, Tan LB; Leeds, UK 4. PREGNANCY OUTCOME IN RENAL TRANSPLANT RECIPIENTS AT ADDENBROOKES HOSPITAL: A 10 YEAR REVIEW Burrell C, Lees C, Daniels I, Somoye G; Cambridge, UK 5. HYPERTENSION IN PREGNANCY: A SURVEY OF CLINICAL PRACTICE TO DOCUMENT CURRENT PRACTICE IN THE UK AND REFINE QUESTIONS FOR A RANDOMIZED CONTROLLED TRIAL (RCT) Churchill D, Duley L, Farrell B; Wolverhampton, UK 6. THE IMPACT OF ACQUIRED THROMBOPHILIA ON MATERNAL AND FETAL WELLBEING IN A LOW-RISK POPULATION Cooley SM, Donnelly J, Walsh T, Geary MP, Gillan J, MacMahon C; Dublin, Ireland 7. FREE MATERNAL DNA IS INCREASED BEFORE 20 WEEKS GESTATION IN WOMEN WHOSE PREGNANCIES ARE SUBSEQUENTLY COMPLICATED BY IUGR BUT NOT PREECLAMPSIA Crowley A, Fitzpatrick P, Sheils O, O’Leary J, O’Herlihy C, Byrne B; Dublin, Ireland 8. IMPACT OF NATIONAL GUIDELINES ON THE MANAGEMENT OF VENOUS THROMBOEMBOLISM IN PREGNANCY Doumouchtsis SK, Tartaglia MA, Thomas GMA, Wilson MP, Thomson AJ; Paisley, UK 9. AN AUDIT ON THE MANAGEMENT OF ANAEMIA IN PREGNANCY IN A LARGE DISTRICT GENERAL HOSPITAL Dua A, Schram CMH, Karunakaran B; Blackburn, UK 10. MECHANISMS OF ACTION OF TNF-a ON ENDOTHELIUM-DEPENDENT RELAXATION IN OMENTAL ARTERIES OBTAINED FROM WOMEN WITH HEALTHY PREGNANCIES Gillham JC, Taggart MJ, Baker PN; Manchester, UK 11. THE QUEENMOTHERS HOSPITAL REVIEW OF PRE-GESTATIONAL DIABETES MELLITUS Hale J, Nicoll AE, Munaza S, Macara LM, Small M, Capaldi B, Cameron AD; Glasgow, UK 12. HEPATITIS B VACCINATION IN AT RISK DRUG USING WOMEN IN PREGNANCY Hyde J, Blunsden V, Irish C; Bristol, UK 13. ALVEOLAR O2 AND CO2 TENSIONS DURING PREGNANCY, MEASURED WITH A NOVEL NON-INVASIVE TECHNIQUE Litos M, Hadjistavrou C, Antsaklis A, Xygakis A, Jordanoglou J; Athens, Greece 14. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) LEVELS IN DIABETIC PREGNANCY: RELATIONS TO NORMAL PREGNANCY AND GLYCAEMIC CONTROL Manderson JG, McClure N, Patterson CC, Hadden DR, Traub AI, McCance DR; Belfast, UK 15. HYPOTHYROIDISM IN PREGNANCY: INCREASE IN THYROXINE DOSE MAY BE TOO LATE USING OUR CURRENT PROTOCOL Masheshwari S, Singh A, Trinder J; Bristol, UK 16. PREGNANCY CARE IN OBESE WOMEN. AN OBSERVATIONAL STUDY IN A DISTRICT GENERAL HOSPITAL Nawar-Youssef MN, Iqbal F, Odukoya OA; Scunthorpe, UK 17. MANAGEMENT OF POST-PARTUM HYPERTENSION (PHT) Ogunnoiki O, Hirsi-Farah S, Gray G, Nelson-Piercy C; London, UK 18. STUDY ON SCREENING FOR GESTATIONAL DIABETES ParveenAS,PeploeD,Bell-ThomasS;Abergavenny,UK 19. DOES THE INTRODUCTION OF A SCREENING PROCESS FOR MENTAL HEALTH PROBLEMS IMPROVE DETECTION OF AT RISK WOMEN IN THE ANTENATAL PERIOD? Scholefield HJ, Hernon M, Topping J; Liverpool, UK 20. MATERNAL OBESITY AND ITS IMPACT ON PREGNANCY OUTCOME Singhal T, Vogiatzi M, Parmeshwaran S, Howarth ES; Leicester, UK 21. RETROSPECTIVE ANALYSIS OF THE OUTCOME OF HIV PREGNANCIES IN A LARGE DISTRICT GENERAL HOSPITAL Sivarajan S, Hooi A, Roy M, Thomas P, Modi M, Howard R, Sahoo S; London, UK 22. PLACENTAL ABCA1 PROTEIN EXPRESSION IN ANTI-PHOSPHOLIPID SYNDROME TetlowN, Albrecht C, Lakasing L, Soumian S, Patel P, Sullivan M, Nicolaides K, Williamson C; London, UK 23. VIRILIZATION IN PREGNANCY ASSOCIATED WITH OVARIAN LEIOMYOMAS Udayasankar V, Moselhi M, Fielding A; Swansea, UK Journal of Obstetrics and Gynaecology (April 2005) Vol. 25, Supplement 1, S27–S31