Journal
BIOCHEMICAL JOURNAL
Volume 390, Issue -, Pages 583-590Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20050294
Keywords
Bacillus subtilis; bifunctional enzyme; chorismate mutase; 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS); Marburg strain; metalloenzyme
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Funding
- NIGMS NIH HHS [GM 53069, R01 GM053069] Funding Source: Medline
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The mono/bifunctional and metallo/non-metallo properties of Bacillus subtilis DAHPS (3-deoxy-D-at-abino-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 K-m(PEP) = 139 +/- 11.4 mu M for phosphoenolpyruvate, K-m(E4P) = 1760 +/- 110 mu M for D-erythrose 4-phosphate, k(cat) = 4.6 +/- 0.1 s(-1) for DAHPS activity and Km chorismate = 850 +/- 97 mu M, k(cat) = 0.41 +/- 0.01 s(-1) for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.
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