Structure of the wild-type TEM-1 beta-lactamase at 1.55 A and the mutant enzyme Ser70Ala at 2.1 A suggest the mode of noncovalent catalysis for the mutant enzyme.

One of the best-studied examples of a class A beta-lactamase is Escherichia coli TEM-1 beta-lactamase. In this class of enzymes, the active-site serine residue takes on the role of a nucleophile and carries out beta-lactam hydrolysis. Here, the structures of the wild-type and the S70G enzyme determined to 1.55 and 2.1 A, respectively, are presented. In contrast to the previously reported 1.8 A structure, the active site of the wild-type enzyme (1.55 A) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X-ray crystal structure of the S70G mutant has an altered Ser130 side-chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild-type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active-site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.

Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase.

Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp(51). The D153E enzyme had an increased k(cat) in the presence of high concentrations of Mg(2+), along with a decreased Mg(2+) affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn(1) site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn(2+), dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.

A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells.

The PI3K/PTEN/Akt signal transduction pathway plays a key role in many tumors. Downstream targets of this pathway include the Forkhead family of transcription factors (FOXO1a, FOXO3a, FOXO4). In PTEN null cells, FOXO1a is inactivated by PI3K-dependent phosphorylation and mislocalization to the cytoplasm, yet still undergoes nucleocytoplasmic shuttling. Since forcible localization of FOXO1a to the nucleus can reverse tumorigenicity of PTEN null cells, a high-content, chemical genetic screen for inhibitors of FOXO1a nuclear export was performed. The compounds detected in the primary screen were retested in secondary assays, and structure-function relationships were identified. Novel general export inhibitors were found that react with CRM1 as well as a number of compounds that inhibit PI3K/Akt signaling, among which are included multiple antagonists of calmodulin signaling.

Altering of the metal specificity of Escherichia coli alkaline phosphatase.

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity.

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.