Nikolaus Amrhein

ABI1 of Arabidopsis is a protein serine/threonine phosphatase highly regulated by the proton and magnesium ion concentration

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, maintenance of seed dormancy, and inhibition of plant growth. All three responses are regulated by the ABI1 gene product. The ABI1 protein (ABI1p) has been characterized as a protein serine/threonine phosphatase of type 2C that is highly affected in its activity by changes in the proton and magnesium ion concentrations. In the ABA‐insensitive mutant abi1 of Arabidopsis thaliana a single amino acid exchange in the primary structure results in both a dominant insensitive phenotype and a strongly reduced protein phosphatase activity in vitro by possibly impairing metal ion coordination.

The UDP-N-acetylglucosamine 1-carboxyvinyl-transferase of Enterobacter cloacae Molecular cloning, sequencing of the gene and overexpression of the enzyme

The UDP‐N‐acetylglucosamine 1‐carboyyvinyltransferase (enol‐pyruvyltransferase, EC 2.5,1.7) which catalyses the first committed step in the biosynthesis of the bacterial cell‐wall peptidoglycan was purified to near homogeneity from Enterobacter cloacae and the NH2‐terminal amino‐acid sequence determined. Using the polymerase chain reaction a 53‐bp DNA fragment was synthesized; this fragment encodes the NH2‐terminal sequence of the enzyme. A clone was then isolated which contained an open reading frame of 1257 bp coding for a protein of 419 amino acids. This protein was overexpressed 100‐fold in transformed Escherichia coli cells and shown to possess the enolpyruvyltransferase activity. The overall amino‐acid sequence of the enolpyruvyltransferase is significantly similar to that of the 5‐enolpyruvylshikimate 3‐phosphate synthase, the only other enzyme known to catalyse the transfer of the enolpyruvate moiety of phosphoenolpyruvate to a substrate.

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH.

Chorismate synthase catalyzes theanti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed “bifunctional.” We have cloned chorismate synthase from the common bread mold Neurospora crassa,expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.

Chorismate Synthase from the Hyperthermophile Thermotoga maritima Combines Thermostability and Increased Rigidity with Catalytic and Spectral Properties Similar to Mesophilic Counterparts

Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a biochemically unique reaction in that it requires reduced FMN as a cofactor. Here we report on the cloning, expression, and characterization of the protein for the first time from an extremophilic organism Thermotoga maritima which is also one of the oldest and most slowly evolving eubacteria. The protein is monofunctional in that it does not have an intrinsic ability to reduce the FMN cofactor and thereby reflecting the nature of the ancestral enzyme. Circular dichroism studies indicate that the melting temperature of the T. maritima protein is above 92u2009°C compared with 54u2009°C for the homologousEscherichia coli protein while analytical ultracentrifugation showed that both proteins have the same quaternary structure. Interestingly, UV-visible spectral studies revealed that the dissociation constants for both oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10-fold, respectively, upon heat treatment of the T. maritima protein. The heat treatment also results in the trapping of the flavin cofactor in an apolar environment, a feature which is enhanced by the presence of the substrate 5-enolpyruvylshikimate 3-phosphate. Nevertheless, stopped-flow spectrophotometric evidence suggests that the mechanism of the T. maritima protein is similar to that of the E. coli protein. In essence, the study shows that T. maritima chorismate synthase exhibits considerably higher rigidity and thermostability while it has conserved features relevant to its catalytic function.

Time‐resolved solid‐state REDOR NMR studies of UDP N‐acetylglucosamine enolpyruvyl transferase

The new method of time‐resolved solid‐state rotational echo double resonance (REDOR) NMR spectroscopy introduced recently by this laboratory has been applied to the enzyme uridine N‐acetylglucosamine (UDP‐NAG) enolpyruvyl transferase (EPT), with the goal of probing the interactions between reactive species and their enzyme active site. The approach has been used in a qualitative fashion with the enzyme‐inhibitor and enzyme‐intermediate complexes of uniformly 15N‐labeled UDP‐NAG EPT, trapped under steady‐state and pre‐steady‐state conditions. A different set of intermolecular interactions between the substrates UDP‐NAG, UDP‐NAG plus 3‐Z‐fluorophosphoenolpyruvate, covalent O‐phosphothioketal, and UDP‐NAG plus phosphoenolpyruvate trapped under time‐resolved conditions (after 50 ms reaction time), and the EPT enzyme active site were observed, and this is contrasted to a similar study of the interactions in a related enzyme, 5‐enolpyruvyl‐shikimate‐3‐phosphate synthase.