Phenylalanine hydroxylase and isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in relationship to the phylogenetic position of Pseudomonas acidovorans (Ps. sp. ATCC 11299a)

The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging. One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads. Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis. This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown. Although P. acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P. acidovorans ATCC 15668 (type strain). This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species. Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either l-tyrosine or l-phenylalanine. Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr. DAHP synthase-tyr is tightly controlled by l-tyrosine with 50% inhibition of activity being achieved at 4.0 M effector. DAHP synthase-phe is inhibited 50% by 40 M l-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol. This two-isozyme pattern of DAHP synthase has not been described previously, although it may prove to be widespread.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - E4P d-erythrose-4-phosphate - PEP phosphoenolpyruvate - DTT dithiothreitol - BSA fraction V bovine serum albumin