Expression, purification, crystallization and preliminary X-ray analysis of the PaaI-like thioesterase SAV0944 from Staphylococcus aureus

Yogesh B. Khandokar, Noelia Roman, Kate M. Smith, Parul Srivastava, Jade K Forwood

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Staphylococcus aureus is the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) from S. aureus subsp. aureus Mu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization of SaPaaI thioesterase from S. aureus are reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24% PEG 4000, 100 mM sodium citrate pH 6.5, 12% 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 Å resolution at the Australian Synchrotron and belonged to space group P1211, with unit-cell parameters a = 44.05, b = 89.05, c = 60.74 Å, β = 100.5°. Initial structure determination and refinement gave an R factor and R free of 17.3 and 22.0%, respectively, confirming a positive solution in obtaining phases using molecular replacement.

Original languageEnglish
Pages (from-to)244-247
Number of pages4
JournalActa Crystallographica. Section F: Structural Biology Communications
Volume70
Issue number2
DOIs
Publication statusPublished - 2014
Externally publishedYes

Keywords

  • hotdog fold
  • phenylacetyl-CoA
  • Staphylococcus aureus
  • thioesterase

Cite this

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title = "Expression, purification, crystallization and preliminary X-ray analysis of the PaaI-like thioesterase SAV0944 from Staphylococcus aureus",
abstract = "Staphylococcus aureus is the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) from S. aureus subsp. aureus Mu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization of SaPaaI thioesterase from S. aureus are reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24{\%} PEG 4000, 100 mM sodium citrate pH 6.5, 12{\%} 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 {\AA} resolution at the Australian Synchrotron and belonged to space group P1211, with unit-cell parameters a = 44.05, b = 89.05, c = 60.74 {\AA}, β = 100.5°. Initial structure determination and refinement gave an R factor and R free of 17.3 and 22.0{\%}, respectively, confirming a positive solution in obtaining phases using molecular replacement.",
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Expression, purification, crystallization and preliminary X-ray analysis of the PaaI-like thioesterase SAV0944 from Staphylococcus aureus. / Khandokar, Yogesh B.; Roman, Noelia; Smith, Kate M.; Srivastava, Parul; Forwood, Jade K.

In: Acta Crystallographica. Section F: Structural Biology Communications, Vol. 70, No. 2, 2014, p. 244-247.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Forwood, Jade K

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