Cyclooxygenase enzymes: catalysis and inhibition.

Scientists working in the field of cyclooxygenase enzymes have witnessed several major advances in the past two years. Crystal structures of fatty acid substrate and prostaglandin product complexes have been elucidated. Elegant site-directed mutagenesis studies have pinpointed the roles of key amino acids within the active site. Together, these results have provided key insights into the overall reaction mechanism. Detailed kinetics, spectroscopic and crystallographic studies have shed new light on the complex mechanism of inhibition of these fascinating enzymes. Finally, novel substrates of cyclooxygenase-2 have been identified.

A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors.

Cyclo-oxygenase (COX) enzymes are the targets for non-steroidal anti-inflammatory drugs (NSAIDs). These drugs demonstrate a variety of inhibitory mechanisms, which include simple competitive, as well as slow binding and irreversible inhibition. In general, most NSAIDs inhibit COX-1 and -2 by similar mechanisms. A unique class of diarylheterocyclic inhibitors has been developed that is highly selective for COX-2 by virtue of distinct inhibitory mechanisms for each isoenzyme. Several of these inhibitors, with varying selectivity, have been utilized to probe the mechanisms of COX inhibition. Results from analysis of both steady-state and time-dependent inhibition were compared. A generalized mechanism for inhibition, consisting of three sequential reversible steps, can account for the various types of kinetic behaviour observed with these inhibitors.

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon.

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 A with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 A resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.

DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution.

BACKGROUND: The study of DNA polymerases in the Pol l family is central to the understanding of DNA replication and repair. DNA polymerases are used in many molecular biology techniques, including PCR, which require a thermostable polymerase. In order to learn about Pol I function and the basis of thermostability, we undertook structural studies of a new thermostable DNA polymerase. RESULTS: A DNA polymerase large, Klenow-like, fragment from a recently identified thermostable strain of Bacillus stearothermophilus (BF) was cloned, sequenced, overexpressed and characterized. Its crystal structure was determined to 2.1 A resolution by the method of multiple isomorphous replacement. CONCLUSIONS: This structure represents the highest resolution view of a Pol I enzyme obtained to date. Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3'-5' proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft.

Linkage of a new mutation in the proteolipid protein (PLP) gene to Pelizaeus-Merzbacher disease (PMD) in a large Finnish kindred.

The purpose of this study was to confirm linkage of the proteolipid protein gene (PLP) and Pelizaeus-Merzbacher disease (PMD). A T-->A transversion in nucleotide pair 35 of exon 4 of PLP was found in a large Finnish kindred with PMD. This mutation results in the substitution Val165-->Glu165. We used a combination of single-strand conformational polymorphism and PCR primer extension to determine the presence or absence of the point mutation in family members. A lod score of 2.6 (theta = 0) was found for linkage of the gene and the disease. We examined 101 unrelated X chromosomes and found none with the transversion. This is the second report of linkage of PMD to a missense mutation in PLP. These findings support the hypothesis that PMD in this family is a result of the missense mutation present in exon 4 of PLP.