The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.

Abstract

The pyridoxal-5-phosphate-dependent enzymes (B6 enzymes) that act on amino acid substrates are of multiple evolutionary origin. The numerous common mechanistic features of B6 enzymes thus are not historical traits passed on from a common ancestor enzyme but rather reflect evolutionary or chemical necessities. Family profile analysis of amino acid sequences supported by comparison of the available three-dimensional (3-D) crystal structures indicates that the B6 enzymes known to date belong to four independent evolutionary lineages of homologous (or more precisely paralogous) proteins, of which the alpha family is by far the largest. The alpha family (with aspartate aminotransferase as the prototype enzyme) includes enzymes that catalyze, with several exceptions, transformations of amino acids in which the covalency changes are limited to the same carbon atom that carries the amino group forming the imine linkage with the coenzyme (i.e., Calpha in most cases). Enzymes of the beta family (tryptophan synthase beta as the prototype enzyme) mainly catalyze replacement and elimination reactions at Cbeta. The D-alanine aminotransferase family and the alanine racemase family are the two other independent lineages, both with relatively few member enzymes. The primordial pyridoxal-5-phosphate-dependent enzymes apparently were regio-specific catalysts that first diverged into reaction-specific enzymes and then specialized for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages. Comparison of sequences from eukaryotic, archebacterial, and eubacterial species indicates that the functional specialization of most B6 enzymes has occurred already in the universal ancestor cell. The cofactor pyridoxal-5-phosphate must have emerged very early in biological evolution; conceivably, organic cofactors and metal ions were the first biological catalysts. In attempts to stimulate particular steps of molecular evolution, oligonucleotide-directed mutagenesis of active-site residues and directed molecular evolution have been applied to change both the substrate and reaction specificity of existent B6 enzymes. Pyridoxal-5-phosphate-dependent catalytic antibodies were elicited with a screening protocol that applied functional selection criteria as they might have been operative in the evolution of protein-assisted pyridoxal catalysis.