We report the first molecular genetic analysis of a pyridoxine 5'-phosphate oxidase, the PdxH gene product of Escherichia coli K-12. Chromosomal insertions in and around pdxH were generated with various transposons, and the resulting phenotypes were characterized. The DNA sequence of pdxH was determined, and the promoters of pdxH and the downstream gene tyrS, which encodes tyrosyl-tRNA synthetase, were mapped by RNase T2 protection assays of chromosomal transcripts. These combined approaches led to the following conclusions: (i) pdxH is transcribed from a sigma 70-type promoter and shares its transcript with tyrS; (ii) tyrS is additionally transcribed from a relatively strong, nonconventional internal promoter that may contain an upstream activating sequence but whose expression is unaffected by a fis mutation; (iii) PdxH oxidase is basic, has a molecular mass of 25,545 Da, and shares striking homology (greater than 40% identity) with the developmentally regulated FprA protein of Myxococcus xanthus; (iv) mild pyridoxal 5'-phosphate limitation of pdxH mutants inhibits cell division and leads to formation of unsegregated nucleoids; (v) E. coli PdxH oxidase is required aerobically and anaerobically, but second-site suppressors that replace pdxH function entirely can be isolated; and (vi) pdxH mutants excrete significant amounts of L-glutamate and a compound, probably alpha-ketoisovalerate, that triggers L-valine inhibition of E. coli K-12 strains. These findings extend earlier observations that pyridoxal 5'-phosphate biosynthetic and aminoacyl-tRNA synthetase genes are often members of complex, multifunctional operons. Our results also show that loss of pdxH function seriously disrupts cellular metabolism in unanticipated ways.