Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity.In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the irnine linkage with the coenzyme. This family was thus named a family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminulevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup 111), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze p-elimination reactions. The p family includes L-and Dserine dehydratase, threonine dehydratase, the /? subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze p-replacement or p-elimination reactions. The y family incorporates 0-succinylhomoserine (thio1)-lyase, 0-acetylhomoserine (thio1)-lyase, and cystathionine y-lyase, which catalyze y-replacement or y-elimination reactions, as well as cystathionine P-lyase.The a and y family might be distantly related with one another, but are clearly not homologous with the / 3 family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the a, /? or y family by the criterion of profile analysis: alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B, enzymes.Pyridoxal-5'-phosphate-dependent enzymes (B, enzymes) catalyze such a wide variety of transformations of amino acids that they are found in no fewer than four out of the total six EC classes of enzymes (Enzyme Nomenclature, 1992). This paper is part of a study on the evolutionary relationships among these multifarious enzymes. Previously, we have found that most if not all aminotransferases constitute a group of homologous proteins . Profile analysis, an algorithm for the detection of distant relationships between amino acid sequences (Gribskov et al., 1990), showed, however, that the evolutionary relationships extend beyond the group of aminotransferases and include other B, enzymes Mehta and Christen, 1994). This study compares the currently known amino acid sequences of B, enzymes and shows that many of them belong to one of th...