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Research article

Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas

Jing Wu, Galina Ya. Sheflyan, Ronald W. Woodard
Biochemical Journal Sep 01, 2005, 390 (2) 583-590; DOI: 10.1042/BJ20050294
Jing Wu
Department of Medicinal Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, U.S.A.
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Galina Ya. Sheflyan
Department of Medicinal Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, U.S.A.
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Ronald W. Woodard
Department of Medicinal Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, U.S.A.
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Abstract

The mono/bifunctional and metallo/non-metallo properties of Bacillus subtilis DAHPS (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) have been controversial for several decades. The present study investigated the DAHPSs from both the B. subtilis parent Marburg strain and the derivative strain 168 in detail and clarified the above two long-standing questions. The DAHPSs from the parent and the derivative 168 strains have identical sequence and are both bifunctional enzymes with a CM (chorismate mutase) activity and a DAHPS activity. The parent strain expresses a second independent monofunctional CM, encoded by aroH, that is highly active, while the 168 strain expresses an aroH containing a single residue mutation (A112V) that is significantly less active thus leading to previous confusion regarding the mono/bifunctionality of DAHPS. Metal analysis showed that B. subtilis DAHPS as isolated contained iron and zinc and is inactivated by dipicolinic acid; the inactive apoenzyme can be reactivated by bivalent metal ions, indicating that the enzyme is a metalloenzyme. The enzyme-bound metal is insensitive to EDTA treatment, leading to the previous conclusion that this DAHPS does not require a metal. The enzyme displays a homotetrameric structure in solution and appears to follow Michaelis–Menten kinetics with KmPEP=139±11.4 μM for phosphoenolpyruvate, KmE4P=1760±110 μM for D-erythrose 4-phosphate, kcat=4.6±0.1 s−1 for DAHPS activity and Kmchorismate=850±97 μM, kcat=0.41±0.01 s−1 for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.

  • Bacillus subtilis
  • bifunctional enzyme
  • chorismate mutase
  • 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS)
  • Marburg strain
  • metalloenzyme

Abbreviations: aroA(Q)168, DAHPS from B. subtilis 168; aroAMarburg, DAHPS from B. subtilis Marburg; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; CM, chorismate mutase; aroH168, CM from B. subtilis 168; aroHMarburg, CM from B. subtilis Marburg; CM-DAHPSBacillus, DAHPSs from both B. subtilis Marburg and 168 strain; DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DPA, dipicolinic acid; E4P, D-erythrose 4-phosphate; ORF, open reading frame; PEP, phosphoenolpyruvate

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September 2005

Volume: 390 Issue: 2

Biochemical Journal: 390 (2)
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Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas
Jing Wu, Galina Ya. Sheflyan, Ronald W. Woodard
Biochemical Journal Sep 2005, 390 (2) 583-590; DOI: 10.1042/BJ20050294
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Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas
Jing Wu, Galina Ya. Sheflyan, Ronald W. Woodard
Biochemical Journal Sep 2005, 390 (2) 583-590; DOI: 10.1042/BJ20050294

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Keywords

Bacillus subtilis
bifunctional enzyme
chorismate mutase
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS)
Marburg strain
metalloenzyme

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