Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas.
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 K(m)(PEP)=139+/-11.4 microM for phosphoenolpyruvate, K(m)(E4P)=1760+/-110 microM for D-erythrose 4-phosphate, kcat=4.6+/-0.1 s(-1) for DAHPS activity and K(m)(chorismate)=850+/-97 microM, kcat=0.41+/-0.01 s(-1) for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.
Wu
Jing
J
Department of Medicinal Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, USA.
Sheflyan
Galina Ya
GY
Woodard
Ronald W
RW
D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
D008670:Metals; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases