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Enzymes that depend on vitamin B6 (and in particular on its metabolically active form, pyridoxal 5'-phosphate, PLP) are of great relevance to biology and medicine, as they catalyze a wide variety of biochemical reactions mainly involving amino acid substrates. Although PLP-dependent enzymes belong to a small number of independent evolutionary lineages, they encompass more than 160 distinct catalytic functions, thus representing a striking example of divergent evolution. The importance and remarkable versatility of these enzymes, as well as the difficulties in their functional classification, create a need for an integrated source of information about them. The B6 database http://bioinformatics.unipr.it/B6db contains documented B6-dependent activities and the relevant protein families, defined as monophyletic groups of sequences possessing the same enzymatic function. One or more families were associated to each of 121 PLP-dependent activities with known sequences. Hidden Markov models (HMMs) were built from family alignments and incorporated in the database. These HMMs can be used for the functional classification of PLP-dependent enzymes in genomic sets of predicted protein sequences. An example of such analyses (a census of human genes coding for PLP-dependent enzymes) is provided here, whereas many more are accessible through the database itself. The B6 database is a curated repository of biochemical and molecular information about an important group of enzymes. This information is logically organized and available for computational analyses, providing a key resource for the identification, classification and comparative analysis of B6-dependent enzymes.

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by thepro-R _p oxygen of phosphate 9 and by N₇ of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The R _p-, but notS _p-, phosphorothioate reduces the cleavage rate by 10³-fold, and the rate can be fully restored by addition of low concentrations of Cd²⁺, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location ∼20 Å from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd²⁺ binds with a K _d of 25 μm in the ground state and a K _d of 2.5 nm in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

The 8–17 deoxyribozyme is a small RNA-cleaving DNA molecule of potential therapeutic interest. Here, the cleavage rates of 16 variants of the 8–17 deoxyribozyme were measured in the presence of different divalent metal ions. Despite the fact that 8–17 was originally selected in vitro for activity in the presence of Mg²⁺ (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262–4266) nearly all the 8–17 variants exhibited substantially higher (up to 20-fold) reaction rates in Ca²⁺ as compared with Mg²⁺. This preference for calcium ions critically depended on the nucleoside residues at two specific positions of the deoxyribozyme core. The Ca²⁺ specificity of 8–17 is strongly reminiscent of the properties of Mg5, an RNA phosphodiester-cleaving deoxyribozyme previously isolated by Faulhammer and Famulok (Faulhammer, D., and Famulok, M. (1996) Angew. Chem. Int. Ed. Engl. 35, 2837–2841). Indeed, analysis of the Mg5 sequence revealed the presence of a complete 8–17 motif, coincident with the conserved region of Mg5. An 8–17 deoxyribozyme modeled after the Mg5 conserved region displayed catalytic features comparable with those reported for the full-length Mg5 deoxyribozyme.