Purification, crystallization and preliminary X‐ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis
01 Mar 2008-Acta Crystallographica Section F-structural Biology and Crystallization Communications (International Union of Crystallography)-Vol. 64, Iss: 3, pp 167-170
TL;DR: Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli and crystallized in two different crystal forms.
Abstract: Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B.
Citations
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TL;DR: The N-terminal domain of Mtb-DapD exhibits a unique architecture, including an interior water-filled channel, which allows access to a magnesium ion located at the 3-fold symmetry axis, which reveals the binding mode(s) of the co-factor and a possible covalent reaction intermediate.
25 citations
TL;DR: The structure and function of the mycobacterial DAP biosynthetic enzymes are reviewed and a need for new antitubercular agents directed against novel targets is identified.
Abstract: Because of an increased emergence of resistance to current antitubercular drugs, there is a need for new antitubercular agents directed against novel targets. Diaminopimelic acid (DAP) biosynthetic enzymes are unique to bacteria and are absent in mammals and provide a rich source of essential targets for antitubercular chemotherapy. Herein, we review the structure and function of the mycobacterial DAP biosynthetic enzymes.
25 citations
TL;DR: A comprehensive analysis involving bottom‐up systems biology approach was applied wherein potential therapeutic targets of Mycobacterium tuberculosis infections are identified and the therapeutic potential of the selected natural products, including Huperzine A, Rosmarinic acid, and Curcumin based ASD inhibitors are revealed.
Abstract: The emergence of multi-drug resistant strains and co-occurrence of tuberculosis with HIV creates a major burden to the human health globally. Failure of primary antibacterial therapy necessitates the identification of new mycobacterial drugs. In this study, a comprehensive analysis involving bottom-up systems biology approach was applied wherein we have identified potential therapeutic targets of Mycobacterium tuberculosis infections. Our study prioritized M. tuberculosis therapeutic targets [aspartate-β-semialdeyhde dehydrogenase (ASD), dihydrodipicolinate reductase and diaminopimelate decarboxylase] based on flux and elementary mode analysis using direct mathematical modeling of the relevant metabolic pathways. Molecular docking and simulation studies of the priority target (i.e., ASD) revealed the therapeutic potential of the selected natural products (Huperzine A, Rosmarinic acid and Curcumin) based ASD inhibitors. The study highlights the crucial role of systems biology in conjunction with molecular interaction (docking) for probing novel leads against an increasingly resistant pathogen, M. tuberculousis. This article is protected by copyright. All rights reserved
17 citations
TL;DR: The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 Å, and at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzymes.
Abstract: The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 A. The monoclinic crystals contain one tetramer exhibiting D2-symmetry in the asymmetric unit. The tetramer exhibits a donut-like structure with a hollow interior. All four active sites are accessible only from the interior of the tetrameric assembly. Small-angle X-ray scattering indicates that in solution the predominant oligomeric species of the protein is a dimer, but also that higher oligomers exist at higher protein concentrations. The observed scattering data are best explained by assuming a dimer–tetramer equilibrium with about 7% tetramers present in solution. Consequently, at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzyme.
15 citations
Cites background from "Purification, crystallization and p..."
...Dihydrodipicolinate synthase is a tetramer [33, 34], dihydrodipicolinate reductase is also a tetramer [15, 35, 36, 37] and succinyldiaminopimelate aminotransferase [38, 39] as well as aspartate semialdehyde dehydrogenase [40] are dimers....
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TL;DR: The structure of ASADH from Mycobacterium tuberculosis H37Rv has been determined and comparison of the two complex structures revealed that the amino acids Glu224 and Arg249 undergo conformational changes upon binding of glycerol.
Abstract: Aspartate-semialdehyde dehydrogenase (Asd; ASADH; EC 1.2.1.11) is the enzyme that lies at the first branch point in the biosynthetic pathway of important amino acids including lysine and methionine and the cell-wall component diaminopimelate (DAP). The enzymatic reaction of ASADH is the reductive dephosphorylation of aspartyl-β-phosphate (ABP) to aspartate β-semialdehyde (ASA). Since the aspartate pathway is absolutely essential for the survival of many microbes and is absent in humans, the enzymes involved in this pathway can be considered to be potential antibacterial drug targets. In this work, the structure of ASADH from Mycobacterium tuberculosis H37Rv (Mtb-ASADH) has been determined in complex with glycerol and sulfate at 2.18 A resolution and in complex with S-methyl-L-cysteine sulfoxide (SMCS) and sulfate at 1.95 A resolution. The overall structure of Mtb-ASADH is similar to those of its orthologues. However, in the Mtb-ASADH-glycerol complex structure the glycerol molecule is noncovalently bound to the active-site residue Cys130, while in the Mtb-ASADH-SMCS complex structure the SMCS (Cys) is covalently linked to Cys130. The Mtb-ASADH-SMCS complex structurally mimics one of the intermediate steps in the proposed mechanism of ASADH enzyme catalysis. Comparison of the two complex structures revealed that the amino acids Glu224 and Arg249 undergo conformational changes upon binding of glycerol. Moreover, the structures reported here may help in the development of species-specific antibacterial drug molecules against human pathogens.
15 citations
Cites background or methods from "Purification, crystallization and p..."
...Crystals of Mtb-ASADH were grown as described previously (Vyas et al., 2008)....
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...The expressed protein was purified using Ni–NTA affinity chromatography followed by size-exclusion chromatography (Vyas et al., 2008)....
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...Soluble expression and purification of the recombinant MtbASADH protein was achieved using an optimized expression and purification protocol (Vyas et al., 2008)....
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...The functional form of MtbASADH has been reported to be a dimer (Vyas et al., 2008)....
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References
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TL;DR: The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
Abstract: Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
31,667 citations
"Purification, crystallization and p..." refers methods in this paper
...The data were indexed and integrated using DENZO (Otwinowski & Minor, 1997) and scaled using SCALEPACK (Otwinowski & Minor, 1997)....
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TL;DR: An analysis of the solvent content of 116 different crystal forms of globular proteins found that in many cases this range will be sufficiently restrictive to enable the probable number of molecules in the crystallographic asymmetric unit to be determined directly from the molecular weight of the protein and the space group and unit cell dimensions of the crystal.
7,857 citations
"Purification, crystallization and p..." refers background in this paper
...…weight of the protein (38 072 Da), the most likely numbers of molecules in the asymmetric unit of the orthorhombic crystal form are four, corresponding to a Matthews parameter VM of 3.33 Å 3 Da 1 and a solvent content of 63% (Matthews, 1968), or six (VM = 2.22 Å 3 Da 1, solvent content 45%)....
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TL;DR: In this article, a method is described which produces sensible estimates of structure factor moduli from intensity observations, whether the latter are positive or negative, and preliminary applications of the method to data from the protein phosphorylase b are summarized.
Abstract: A method is described which produces sensible estimates of structure factor moduli from intensity observations, whether the latter are positive or negative. Preliminary applications of the method to data from the protein phosphorylase b are summarized.
954 citations
"Purification, crystallization and p..." refers methods in this paper
...Intensities were converted to structurefactor amplitudes using the program TRUNCATE (French & Wilson, 1978; Collaborative Computational Project, Number 4, 1994)....
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TL;DR: In this paper, global indicators of the quality of diffraction data are presented and discussed, and are evaluated in terms of their performance with respect to various tasks, based on the results obtained, it is suggested that some of the conventional indicators still in use in the crystallographic community should be abandoned, such as the nominal resolution dmin or the merging R factor Rmerge, and replaced by more objective and more meaningful numbers, including the effective optical resolution deff,opt and the redundancy-independent merging R Factor Rr.i.m.
Abstract: Global indicators of the quality of diffraction data are presented and discussed, and are evaluated in terms of their performance with respect to various tasks. Based on the results obtained, it is suggested that some of the conventional indicators still in use in the crystallographic community should be abandoned, such as the nominal resolution dmin or the merging R factor Rmerge, and replaced by more objective and more meaningful numbers, such as the effective optical resolution deff,opt and the redundancy-independent merging R factor Rr.i.m.. Furthermore, it is recommended that the precision-indicating merging R factor Rp.i.m. should be reported with every diffraction data set published, because it describes the precision of the averaged measurements, which are the quantities normally used in crystallography as observables.
668 citations
"Purification, crystallization and p..." refers methods in this paper
...The redundancyindependent merging R factor Rr.i.m. as well as the precisionindicating merging R factor Rp.i.m. (Weiss, 2001) were calculated using the program RMERGE (available from http://www. embl-hamburg.de/~msweiss/projects/msw_qual.html or from MSW upon request)....
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...=P hkl P i IiðhklÞ, with N being the number of times a given reflection hkl was observed; precision-indicating merging R factor Rp.i.m. = 100 P hkl ½1=ðN 1Þ 1=2 P i jIiðhklÞ hIðhklÞij= P hkl P i IiðhklÞ (Weiss, 2001)....
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TL;DR: The asd mutants of Salmonella typhimurium have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP, which has allowed the development of a balanced-lethal system for the expression of heterologous antigens in vaccine strains using vectors containing the wild-type asd gene from Streptococcus mutans and asd mutantSalmonella hosts.
304 citations
"Purification, crystallization and p..." refers background in this paper
...The absence of the two pathways in humans and the absolute requirement for DAP in bacteria (Galan et al., 1990; Pavelka & Jacobs, 1996; Harb & Kwaik, 1998) and the importance of these four amino acids for protein synthesis make the enzymes in these pathways attractive targets for antibacterial drug…...
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...Studies on Asd from Salmonella typhimurium (Galan et al., 1990) and Legionella pneumophila (Harb & Kwaik, 1998) have demonstrated that perturbations of the asd gene encoding Asd are lethal to microbes....
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