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Title: Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved

Abstract

A method is presented that allowed the diffraction limit of crystals of the secreted chorismate mutase from M. tuberculosis to be improved from approximately 3.5 to 1.3 Å. To obtain large well diffracting crystals, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min. Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ{sub γ} subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000 ▶), Protein Sci.9, 755–757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 Å resolution. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 Å, βmore » = 104.5°. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.« less

Authors:
 [1];  [2];  [1];  [3];  [1];  [2]; ;  [3];  [4]
  1. Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg (Sweden)
  2. (Norway)
  3. Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich (Switzerland)
  4. (Sweden)
Publication Date:
OSTI Identifier:
22356324
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 5; Other Information: PMCID: PMC2219981; PMID: 16682771; PUBLISHER-ID: pu5123; OAI: oai:pubmedcentral.nih.gov:2219981; Copyright (c) International Union of Crystallography 2006; This is an open-access article distributed under the terms described at http://journals.iucr.org/services/termsofuse.html.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CONVERSION; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; DIMERS; NUCLEATION; OPTIMIZATION; PROTEINS; RESOLUTION; SPACE GROUPS

Citation Formats

Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Dey, Raja, Sasso, Severin, Ökvist, Mats, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Ramakrishnan, Chandra, Kast, Peter, and Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg. Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106012036.
Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Dey, Raja, Sasso, Severin, Ökvist, Mats, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Ramakrishnan, Chandra, Kast, Peter, & Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg. Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved. United Kingdom. doi:10.1107/S1744309106012036.
Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Dey, Raja, Sasso, Severin, Ökvist, Mats, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Ramakrishnan, Chandra, Kast, Peter, and Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg. Mon . "Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved". United Kingdom. doi:10.1107/S1744309106012036.
@article{osti_22356324,
title = {Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved},
author = {Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no and Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo and Dey, Raja and Sasso, Severin and Ökvist, Mats and Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo and Ramakrishnan, Chandra and Kast, Peter and Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg},
abstractNote = {A method is presented that allowed the diffraction limit of crystals of the secreted chorismate mutase from M. tuberculosis to be improved from approximately 3.5 to 1.3 Å. To obtain large well diffracting crystals, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min. Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ{sub γ} subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000 ▶), Protein Sci.9, 755–757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 Å resolution. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 Å, β = 104.5°. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.},
doi = {10.1107/S1744309106012036},
journal = {Acta Crystallographica. Section F},
number = Pt 5,
volume = 62,
place = {United Kingdom},
year = {Mon May 01 00:00:00 EDT 2006},
month = {Mon May 01 00:00:00 EDT 2006}
}
  • Chorismate mutase from M. tuberculosis has been crystallized. Preliminary X-ray crystallographic studies reveal the occurrence of a dimeric molecule in the crystal asymmetric unit. Chorismate mutase catalyzes the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine in bacteria, fungi and higher plants. The recent re-annotation of the Mycobacterium tuberculosis genome has revealed the presence of a duplicate set of genes coding for chorismate mutase. The mycobacterial gene Rv1885c bears <20% sequence homology to other bacterial chorismate mutases, thus serving as a potential target for the development of inhibitors specific to the pathogen. The M.more » tuberculosis chorismate mutase was crystallized in space group C2 and the crystals diffracted to a resolution of 2.2 Å. Matthews coefficient and self-rotation function calculations revealed the presence of two monomers in the asymmetric unit.« less
  • The Rv0948c gene from Mycobacterium tuberculosis H{sub 37}R{sub v} encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 5.5 {+-} 0.2 s{sup -1} and a K{sub m} of 1500 {+-} 100 {mu}m at 37 C and pH 7.5. The 2.0 {angstrom} X-ray structure shows that 90-MtCM is an all {alpha}-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequencemore » alignment shows that the C-terminus helix 3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k{sub cat}. Hence, 90-MtCM belongs to a subfamily of {alpha}-helical AroQ CMs termed AroQ{sub {delta}}. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 70 {+-} 5 s{sup -1} and K{sub m} of 500 {+-} 50 {mu}m at 37 C and pH 7.5. The 2.1 {angstrom} X-ray structure shows that *YpCM is an all {alpha}-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ{sub {gamma}} class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.« less
  • The Rv0948c gene from Mycobacterium tuberculosis H{sub 37}R{sub v} encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 5.5 {+-} 0.2 s{sup -1} and a K{sub m} of 1500 {+-} 100 {micro}m at 37 C and pH 7.5. The 2.0 {angstrom} X-ray structure shows that 90-MtCM is an all {alpha}-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequencemore » alignment shows that the C-terminus helix 3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k{sub cat}. Hence, 90-MtCM belongs to a subfamily of {alpha}-helical AroQ CMs termed AroQ{delta}. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 70 {+-} 5 s{sup -1} and Km of 500 {+-} 50 {micro}m at 37 C and pH 7.5. The 2.1 {angstrom} X-ray structure shows that *YpCM is an all {alpha}-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ{gamma} class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.« less
  • Pantothenate kinase, the first enzyme of the universal coenzyme A biosynthetic pathway, from M. tuberculosis H37Rv has been cloned, expressed, purified and X-ray analysed in two different crystal forms. Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B{sub 5}) to 4′-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3{sub 1}21. Two complete data sets of resolution 2.5 Å (form I) and 2.9more » Å (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 Å and a = b = 107.63, c = 89.85 Å, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement.« less
  • Crystals of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from M. tuberculosis have been grown and a native data set has been collected to a maximum resolution of 2.5 Å. The enzymes of the shikimate pathway are attractive targets for new-generation antimicrobial agents. The first step of this pathway is catalysed by 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAH7P) synthase and involves the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAH7P. DAH7P synthases have been classified into two apparently evolutionarily unrelated types and whereas structural data have been obtained for the type I DAH7P synthases, no structural information is available for their type II counterparts. The typemore » II DAH7P synthase from Mycobacterium tuberculosis was co-expressed as native and selenomethionine-substituted protein with the Escherichia coli chaperonins GroEL and GroES in E. coli, purified and crystallized. Native crystals of M. tuberculosis DAH7P synthase belong to space group P3{sub 1}21 or P3{sub 2}21 and diffract to 2.5 Å, with unit-cell parameters a = b = 203.61, c = 66.39 Å. There are either two or three molecules in the asymmetric unit. Multiwavelength anomalous diffraction (MAD) phasing using selenomethionine-substituted protein is currently under way.« less