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

AFFINE MODIFICATION OF RESTRICTION ENDONUCLEASES AND DNA-METHYLTRANSFERASES BY SUBSTRATE ANALOGS AS INVESTIGATION METHOD OF THESE ENZYMES

The methods of electrophoretic separation in three variants (inhibition in gel, albuvinous and nucleic phoresises) and methods of the high-effective liquid chromatography have been used. The mechanisms for conformation changes of the restriction endonuclease complexes with substrates under action of the cofactor have been proposed firstly, the covalent enzyme adducts with DNA with monosubstituted pyrophosphate internucleotide bond have been made. The optimal conditions of affine protein modification by the NA analogs have been discovered, the optimal scheme for extraction of the oligonucleotidopeptide - trypsinolysis products of albuminous-nucleic conjugate has been developedAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with K(m)=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and k(cat)=5.9 s(−1) at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs

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