In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.Recent work in our laboratory with the filamentous, phytopathogenic fungus Cercospora nicotianae revealed that a highly conserved group of gene homologues found in eubacteria, archaebacteria, fungi, and plants play a role in a divergent pyridoxine (vitamin B 6 ) biosynthesis pathway (8). PDX1 was originally identified as a gene required for resistance of this fungus to a singlet-oxygen-generating toxin, cercosporin, which it produces to parasitize plants (9, 10). During characterization of this gene, however, we discovered that it rescued both C. nicotianae and Aspergillus flavus pyridoxine auxotrophs to prototrophy (8). This observation was subsequently confirmed in Aspergillus nidulans, in which a PDX1 homologue, PYROA, also rescued pyridoxine auxotrophy (24). Interestingly, despite this direct evidence for the involvement of PDX1 homologues in pyridoxine synthesis, PDX1 shows no homology to any of the known Escherichia coli pyridoxine biosynthesis genes or to any gene in the completely sequenced E. coli genome. Database analysis determined that organisms with homologues to the E. coli genes (some eubacteria) lacked PDX1 homologues and that organisms with PDX1 homologues (other eubacteria, archaebacteria, fungi, and plants) lacked homologues to the E. coli genes. These data suggested that a divergence in the pyridoxine synthesis pathway occurred sometime during the evolution of the eubacteria (8).The advent of genomic and other large-scale sequencing projects allows homology comparisons on an organismal level. Saccharomyces cerevisiae contains three unlinked PDX1 homologues, one of which (SNZ1, for snooze) was extensively studied because its expression increases dramatically during stationary phase (5). Analyses by Galperin and Koonin (12) uncovered that PDX1-containing organisms also contained a copy of a second homologous gene and that three of these organisms (S. cerevi...