http://hdl.handle.net/10027/7545 Sat, 25 May 2013 04:44:02 GMT 2013-05-25T04:44:02Z http://hdl.handle.net/10027/8485 Byrsonima fagifolia Niedenzu Apolar Compounds with Antitubercular Activity Higuchi, C.; Sannomiya, M.; Pavan, F.; Leite, S.; Sato, D.; Franzblau, S.; Sacramento, L.V.; Vilegas, W.; Leite, C. Bioassay-guided fractionation of the chloroform extract of Byrsonima fagifolia leaves led to the isolation of active antitubercular compounds alkane dotriacontane (Minimal Inhibitory Concentration-MIC, 62.5 mu g mL(-1)), triterpenoids as bassic acid (MIC = 2.5 mu g mL(-1)), alpha-amyrin acetate (MIC = 62.5 mu g mL(-1)), a mixture of lupeol, alpha- and beta-amyrin (MIC = 31.5 mu g mL(-1)) and a mixture of lupeol, and acetates of alpha- and beta-amyrin (MIC = 31.5 mu g mL(-1)). The antimycobacterial activity was determined by the Microplate Alamar Blue Assay (MABA) and the structures of promising compounds were determined by spectroscopic analysis. This investigation constitutes the first report of a chemical and antitubercular study of apolar compounds from B. fagifolia Niedenzu (IK). Copyright © 2011 C. T. Higuchi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/ecam/nen077 Sat, 01 Jan 2011 06:00:00 GMT http://hdl.handle.net/10027/8485 2011-01-01T06:00:00Z http://hdl.handle.net/10027/8192 Crystallization and preliminary X-ray characterization of the glpX-encoded class II fructose-1,6-bisphosphatase from Mycobacterium tuberculosis Gutka, Hiten J.; Franzblau, Scott G.; Movahedzadeh, Farahnaz; Abad-Zapatero, Cele Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7 Å and belonged to the hexagonal space group P6122, with unit-cell parameters a = b = 131.3, c = 143.2 Å. The structure has been solved by molecular replacement and is currently undergoing refinement. The original version is available through International Union of Crystallography at DOI: 10.1107/S1744309111014722. Wed, 01 Jun 2011 05:00:00 GMT http://hdl.handle.net/10027/8192 2011-06-01T05:00:00Z http://hdl.handle.net/10027/8172 A Two-Step Strategy for the Complementation of M. Tuberculosis Mutants Movahedzadeh, Farahnaz; Frita, Rosangela; Gutka, Hiten J. The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The original version is available through the Brazilian Genetics Society at DOI: 10.1590/S1415-47572011000200020. Sat, 01 Jan 2011 06:00:00 GMT http://hdl.handle.net/10027/8172 2011-01-01T06:00:00Z http://hdl.handle.net/10027/7706 glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II Gutka, Hiten J.; Rukseree, Kamolchanok; Wheeler, Paul R.; Franzblau, Scott G.; Movahedzadeh, Farahnaz The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6- bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size 2 exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis–Menten kinetics with an apparent km = 44 μM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme. Post print version of article may differ from published version. The original publication is available at springerlink.com; DOI: 10.1007/s12010-011-9219-x. Thu, 31 Mar 2011 05:00:00 GMT http://hdl.handle.net/10027/7706 2011-03-31T05:00:00Z