Probing the potential metal binding site in Escherichia coli 3‐deoxy‐d‐arabino‐heptulosonate 7‐phosphate synthase (phenylalanine‐sensitive)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116945/1/feb2s0014579398015452.pd

Kinetic and Mutagenic Evidence for the Role of Histidine Residues in the Lycopersicon esculentum 1-Aminocyclopropane-1-carboxylic acid Oxidase

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M −1 min −1 . The pH–inactivation rate data imply the involvement of an amino acid residue with a p K value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45083/1/10930_2004_Article_409396.pd

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 μM for phosphoenolpyruvate, K(m)(E4P)=1760±110 μM for D-erythrose 4-phosphate, k(cat)=4.6±0.1 s(−1) for DAHPS activity and K(m)(chorismate)=850±97 μM, k(cat)=0.41±0.01 s(−1) for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate