Ten years of experience with molecular class-specific information systems (MCSIS) such as with the hand-curated G protein-coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein-related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes "all" available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three-dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA-diagnostics, drug design, and so forth.

The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in L-galactono-␥-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 3 Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (k ox ‫؍‬ 3.5 ؋ 10 5 M ؊1 s ؊1 ), similar to most other flavoprotein oxidases. The Ala-113 3 Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.The flavoenzyme L-galactono-␥-lactone dehydrogenase (GALDH; 2 EC 1.3.2.3) catalyzes the terminal step in the biosynthesis of vitamin C (L-ascorbate) in plants. Besides producing this essential nutrient, GALDH is required for the accumulation of plant respiratory complex I (1). GALDH is an aldonolactone oxidoreductase that belongs to the vanillyl-alcohol oxidase (VAO) flavoprotein family (2). Members of this family share a two-domain folding topology with a conserved FAD binding domain and a cap domain that defines the substrate specificity (3). VAO family members include enzymes involved in carbohydrate metabolism and lignin degradation and enzymes that participate in the synthesis of antibiotics and alkaloids (4). Most VAO members contain a covalently tethered FAD and act as oxidases that use molecular oxygen to reoxidize the flavin, resulting in the production of hydrogen peroxide. In contrast to related aldonolactone oxidoreductases like L-gulono-␥-lactone oxidase from animals (5) and D-arabinono-␥-lactone oxidase from yeast (6), GALDH reacts poorly with molecular oxygen and contains non-covalently bound FAD (7). No crystal structure is available for the aldonolactone oxidoreductase subfamily, and little is known about the nature of the active site and the catalytic mechanism.GALDH is localized in the mitochondrial intermembrane space, where it feeds electrons into the respiratory chain. Its subcellular localization could provide a rationale why GALDH is a dehydrogenase and not, like related enzymes, an oxidase. The latter activity would result in high levels of mitochondrial hydrogen peroxide that promote GALDH inactivation (8) and induce aging, senescence, and cell death (9, 10). Furthermore, i...

Correlated mutation analyses (CMA) on multiple sequence alignments are widely used for the prediction of the function of amino acids. The accuracy of CMA-based predictions is mainly determined by the number of sequences, by their evolutionary distances, and by the quality of the alignments. These criteria are best met in structure-based sequence alignments of large super-families. So far, CMA-techniques have mainly been employed to study the receptor interactions. The present work shows how a novel CMA tool, called Comulator, can be used to determine networks of functionally related residues in enzymes. These analyses provide leads for protein engineering studies that are directed towards modification of enzyme specificity or activity. As proof of concept, Comulator has been applied to four enzyme super-families: the isocitrate lyase/phoshoenol-pyruvate mutase super-family, the hexokinase super-family, the RmlC-like cupin super-family, and the FAD-linked oxidases super-family. In each of those cases networks of functionally related residue positions were discovered that upon mutation influenced enzyme specificity and/or activity as predicted. We conclude that CMA is a powerful tool for redesigning enzyme activity and selectivity.

The NucleaRDB is a Molecular Class-Specific Information System that collects, combines, validates and disseminates large amounts of heterogeneous data on nuclear hormone receptors. It contains both experimental and computationally derived data. The data and knowledge present in the NucleaRDB can be accessed using a number of different interactive and programmatic methods and query systems. A nuclear hormone receptor-specific PDF reader interface is available that can integrate the contents of the NucleaRDB with full-text scientific articles. The NucleaRDB is freely available at http://www.receptors.org/nucleardb.