Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

The rapid expansion of the amount of genomic and structural data has provided many examples of enzymes with evolutionarily related active sites that catalyze different reactions. Functional comparisons of these active sites can provide insight into the origins of the enormous catalytic proficiency of enzymes and the evolutionary changes that can lead to different enzyme activities. The alkaline phosphatase (AP) superfamily is an ideal system to use in making such comparisons given the extensive data available on both nonenzymatic and enzymatic phosphoryl transfer reactions. Some superfamily members, such as AP itself, preferentially hydrolyze phosphate monoesters, whereas others, such as nucleotide pyrophosphatase/ phosphodiesterase (NPP), preferentially hydrolyze phosphate diesters. We have measured rate constants for NPP-catalyzed hydrolysis of phosphate diesters and monoesters. NPP preferentially catalyzes diester hydrolysis by factors of 10 2 -10 6 , depending on the identity of the diester substrate. To identify features of the NPP active site that could lead to preferential phosphate diester hydrolysis, we have determined the structure of NPP in the absence of ligands and in complexes with vanadate and AMP. Comparisons to existing structures of AP reveal bimetallo cores that are structurally indistinguishable, but there are several distinct structural features outside of the conserved bimetallo site. The structural and functional data together suggest that some of these distinct functional groups provide specific substrate binding interactions, whereas others tune the properties of the bimetallo active site itself to discriminate between phosphate diester and monoester substrates.Enzymes in the alkaline phosphatase (AP) 1 superfamily catalyze a wide variety of phosphoryl and sulfuryl transfer reactions (1). Members of this superfamily include phosphatases, phosphodiesterases, phosphoglycerate mutases, phosphopentomutases, and sulfatases. AP itself preferentially catalyzes phosphate monoester hydrolysis, presumably to harvest phosphate for nucleic acids and metabolites, and its structural and functional properties have been extensively studied (2). The nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes are members of the AP superfamily that catalyze phosphate diester hydrolysis (1,3,4). In eukaryotes, these enzymes are found at cell surfaces, either as transmembrane proteins or secreted, and hydrolyze extracellular phosphate diesters to affect a variety of biological processes (5, 6).Although no experimental structural information was previously available for NPP, a model for the NPP active site that is strikingly similar to the AP active site was proposed on the basis of sequence comparisons and homology modeling. The model consists of two active site metal ions, six conserved metal ligands, and a Thr residue positioned in a manner analogous to that of a Ser residue in AP (Scheme 1) (3). In AP-catalyzed phosphate monoester hydrolysis reactions, the Ser alkoxide displaces the leaving group...

Macromolecular visualization is hampered by the fragmented set of available programs and the lack of cooperativity among them. The amount of visual information required for robust structural analysis is relatively dif®cult to generate and rarely allows further high-quality three-dimensional graphic rendering. Here, a modi®cation of MolScript [Kraulis (1991). J. Appl. Cryst. 24, 946±950] is presented which contains the capability of the original MolScript, the ability to carry out a majority of the options available in most other crystallographic visualization packages, as well as several new features of its own. POVScript+ (currently version 1.62) allows anisotropic displacement ellipsoid rendering (read in as a second-rank tensor from a PDB ®le), electrondensity polygonization (in several formats derived from a`marching tetrahedra' approach), volumetric rendering of electron density and GRASP/MSMS surface-map input/output. Finally, POVRay output is supported (via a modi®ed version of PovScript) to generate high-quality renderings that are easily modi®ed for any of a number of purposes (e.g. animations or altered textures). POVScript+ provides a marked increase in the amount of structural and atomic detail possible, while still allowing a straightforward means of generating this information.